Steric Mass Action Research Articles

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography.

The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities.

Structural characteristics of low-molecular-mass displacers for cation-exchange chromatography: II. Role of the stationary phase

The relative efficacy of a variety of low-molecular-mass displacers was examined on three different stationary phase materials. Several homologous series of displacer molecules were evaluated on these ion- exchange resins using a displacer ranking plot based on the steric mass action model. The results demonstrate that while aromaticity and hydrophobicity can play a significant role in the affinity of displacer molecules on polymethacrylate based and hydrophilized polystyrene–divinylbenzene based materials, this effect is much less pronounced on an agarose based resin. The work presented in this paper demonstrates that different structural features of low-molecular-mass displacers can dominate their affinity on various stationary phase materials employed and provides rules of thumb for the design of high affinity, low-molecular-mass displacers for a variety of commercial cation-exchange materials.

Displacement chromatography using the UNO continuous bed column as a stationary phase

Displacement separations of biopolymers are even more restricted in regard to the upper applicable limit of the flow rate than elution chromatographic ones, when conventional columns packed with porous particles are used. Bio-Rad has recently introduced a new column type, the UNO column, which contrary to conventional HPLC columns is not packed with particles but consists of a continuous porous polymer rod. The plate height of this continuous bed column was found to be nearly independent of the flow rate within the investigated range (0.01-4.5 mL/min, 1.56-701.6 cm/h). The strong ion exchanger columns UNO Q1 (7 x 35 mm, 1.3 mL) and UNO Q6 (12 x 53 mm, 6 mL) were investigated as stationary phases for protein displacement chromatography at elevated flow rates. With the UNO column, displacement separations of alpha-lactalbumin and beta-lactoglobulin (displacer: poly(acrylic acid), Mw 5100) were still possible at flow rates that were 1 order of magnitude higher than those applicable to conventional columns of similar dimensions. In fact the flow rate was limited by the necessity to collect the protein-containing fraction fast enough for an adequate monitoring of the separation rather than by a loss in resolution. While the UNO Q1 column did not allow for the full development of the displacement train, 50 mg of protein could be separated using the UNO Q 6 column. Recoveries were well over 95% in this case. More than 50% of the alpha-lactalbumin was collected in pure and concentrated form (concentration by a factor of 2). The steric mass action model was used to optimize the displacer concentration.

Synthesis and Characterization of High-Affinity, Low Molecular Weight Displacers for Cation-Exchange Chromatography

This paper describes the synthesis and characterization of novel low molecular weight displacers for protein purification in cation-exchange systems. A series of displacers based on the pentaerythritol geometry are examined to identify high-affinity, low molecular weight displacers. The steric mass action (SMA) model is employed to evaluate the relative affinities of these displacers. A linear gradient method is described for the measurement of SMA model parameters of molecules. This work demonstrates that, in addition to the number of charges on a displacer molecule, the presence of aromatic moieties can have a profound effect on affinity. In particular, the placement of aromatic groups near the surface of a molecule is shown to result in significant increases in displacer efficacy. Finally, the efficacy of these displacers is examined in displacement experiments using a model protein. These results indicate that nonspecific interactions can play a major role in determining the affinity of a molecule for ion-exchange systems.

Selective displacement chromatography of proteins

In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers are dependent on both mobile phase salt and displacer concentration. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the product(s) of interest can be selectively displaced while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train, and the high affinity impurities either retained or desorbed in the displacer zone. This type of displacement combines the operational advantages of step gradient and the high resolution inherent in a true displacement process, in a single operation. Theoretical expressions are presented for establishing selective displacement operating conditions (initial salt concentration, displacer concentration) based on the Steric Mass Action parameters of the displacer and the linear Steric Mass Action parameters of the feed proteins. Experimental results are presented to elucidate the concept of selective displacement in both cation and anion exchange systems. A mixture of alpha-lactalbumin and beta-lactoglobulin A and B has been used for anion-exchange systems; a four-protein mixture consisting of ribonuclease B, bovine and horse heart cytochrome c, and lysozyme has been employed in cation exchange systems. This article also demonstrates that on-line monitoring can be readily employed for the selective displacement process, thus facilitating the scale-up and control of the process. This work sets the stage for the development of robust large scale high resolution separations using selective displacement chromatography. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 119-129, 1997.

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.

Combination of the steric mass action and non-ideal surface solution models for overload protein ion-exchange chromatography

A model has been developed for protein ion-exchange equilibria under overloaded chromatographic conditions. This model combines the steric mass action model with the non-ideal surface solution model and accounts for steric hindrance and nearest neighbor interactions between protein-protein, protein-salt and salt-salt in a single formalism. Using the model on protein ion-exchange data, the importance of obtaining ion-exchange heat measurements in addition to adsorption isotherms has been demonstrated. It has been shown that small inaccuracies in the heat of ion-exchange data can lead to large differences in predictions of elution behavior. Additionally, it has also been shown that salt-salt interactions on the surface can strongly influence protein adsorption and that in the presence of these interactions, the shape and orientation of the molecule on the surface are important.

Modeling gradient elution of proteins in ion‐exchange chromatography

AbstractThe steric mass action (SMA) model of ion exchange is employed in concert with appropriate mass balance equations to describe the behavior of a concentrated band of protein under gradient‐elution conditions. A method of characteristics approach is employed to solve the model equations rapidly. The model is shown to accurately predict the gradient elution of the protein cytochrome C in a strong cation‐exchange column. This work constitutes an advance over previously available models as it allows engineers to rapidly consider the effects of nonlinear adsorption in gradient‐elution chromatography using a spreadsheet. Further, it provides significant physical insight into the process of gradient elution for preparative and large‐scale separations.

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.

Optimization of preparative ion-exchange chromatography of proteins: linear gradient separations

In this study, the Steric Mass Action (SMA) model of ion exchange is employed in concert with appropriate mass balance equations to predict the separation performance of preparative ion-exchange chromatography. The model is able to accurately predict linear gradient separations of the proteins α-chymotrypsinogen A, cytochrome c, and lysozyme under overloaded conditions. The optimization behavior of preparative ion-exchange chromatography is examined under conditions of baseline resolution and induced sample displacement. This work demonstrates that a simple iterative procedure can be employed to establish optimal gradient conditions for preparative ion-exchange chromatography of proteins. The results also indicate that under appropriate conditions, sample displacement can be employed to dramatically improve the production rate with minor losses in product yield or purity. Linear gradient separations with sample displacement are also less sensitive to the adsorption properties of the feed stream than baseline resolved separations, resulting in simplified methods development.

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.

Transient profiles in ion‐exchange displacement chromatography

AbstractA theoretical and experimental study of displacement development in cation‐exchange displacement chromatography is carried out. Simulated displacement chromatograms are generated using a numericl model of displacement chromatography, which employs the steric mass action (SMA) formalism to describe the multicomponent nonlinear adsorption of proteins and polyelectrolytes. Simulated profiles of transient displacement chromatography are compared with experimentla displacement over a ranve of operating conditions, and the model accurately predicts transient displacement behaviour. The success of the model indicates that the SMA formalism is an appropriate tool for prechromatograms indicate that significantly higher throughputs can be achieved by operating these protein displacement systems under nondeveloped conditions. In addition, displacement development proceeds faster at lower salt concentration, indicating that a reduction of the displacement systems. This work sets the stage for the optimization of protien displacement chromatography.

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.

Modeling non-linear elution of proteins in ion-exchange chromatography

The problem of non-linear elution of a band of protein in isocratic ion-exchange chromatography leads to a pair of coupled non-linear partial differential equations. The equilibrium may be modeled using the Steric Mass Action (SMA) model of ion exchange, which treats both the salt dependence of protein adsorption and the steric shielding present under non-linear conditions. Neglecting axial dispersion, a model of ideal chromatography is formulated that may be solved by the method of characteristics. The predictions of this relatively simple model are shown to agree with experimental results concerning the non-linear elution of cytochrome c in a strong cation-exchange column. Of particular interest is the existence of two plateaus in the solution of this problem for large injection volumes. While this result cannot be understood or predicted on the basis of the traditional Langmuir isotherm or other currently available descriptions of adsorption, the chromatographic model presented in this work makes this otherwise anomalous result clear. Further, the use of such a model during parameter estimation is discussed.

Investigation of displacer equilibrium properties and mobile phase operating conditions in ion-exchange displacement chromatography

One of the major advantages of displacement chromatography is the simultaneous concentration and purification that can be effected during the process. The steric mass-action model of non-linear ion-exchange chromatography is employed to investigate the affects of mobile phase salt concentration and displacer equilibrium properties on the concentration of proteins in isotachic displacement zones. The results indicate that the salt microenvironment in displacement zones plays a major role in determining the concentration of the displaced proteins. The parameters which affect the salt microenvironment are both the initial salt concentration in the carrier as well as the induced gradient produced during the displacement process. For a given breakthrough volume, the induced gradient is determined by the ratio of the steric factor to the characteristic charge of the displacer. These results indicate that the use of relatively low salt concentrations in the carrier along with displacers with relatively high steric factor to characteristic charge ratios will produce significant concentration of the proteins during the purification process.

Gibbs free energy of adsorption for biomolecules in ion-exchange systems

The Gibbs free energy of adsorption ( ΔG ads 0) was estimated for several amino acids, peptides and proteins in a cation-exchange system. Using the steric mass action formalism that describes biomolecular adsorption in ion-exchange systems, the ΔG ads 0 was chromatographically determined under infinitely dilute conditions. The ΔG ads 0 measured for seven globular proteins ranged from −5.7 to −13.9 kcal/mol. The average bond energy (defined as ΔG ads 0 divided by the number of bonds formed between the protein and the surface) for these proteins varied from −1.1 to −1.7 kcal/mol. These bond energies were found to be comparable to the bond energies for lysine and arginine (−1.1 and −1.5 kcal/mol, respectively), the amino acids which primarily contribute to the cation-exchange of proteins. In contrast, an elevated average bond energy of −2.6 kcal/mol was observed for two peptides and protamine (a polypeptide) suggesting that synergistic binding may play a role for unstructured macromolecules, but not for globular proteins.

Characterization of non-linear adsorption properties of dextran-based polyelectrolyte displacers in ion-exchange systems

Experimental studies were carried out on the non-linear adsorption properties of dextran-based polyelectrolytes in anion- and cation-exchange chromatographic systems. By monitoring both the induced salt gradients and sequential breakthrough fronts, parameters were determined for use in a Steric Mass Action (SMA) model of non-linear ion-exchange chromatography. These parameters include: total ion capacity of the columns, characteristic charge, steric factor, equilibrium constant, and maximum adsorptive capacity for each of the polyelectrolytes. In addition the number of functional groups were determined by elemental analysis. The values of the SMA parameters were found to be independent of salt and polyelectrolyte bulk phase compositions. Parameters were also determined for a variety of proteins. Experimental isotherms for the polyelectrolytes and proteins were compared with those simulated by the SMA model. Finally, the implications of polyelectrolyte adsorption properties with respect to their ability to act as efficient displacers in ion-exchange displacement systems are discussed.

Ion-exchange displacement chromatography of proteins: Dextran-based polyelectrolytes as high affinity displacerss

Dextran-based polyelectrolyte displacers were successfully employed for the displacement purification of proteins in ion-exchange displacement systems. The effect of molecular mass was investigated by examining the efficacy of DEAE-dextran and dextran sulfate displacers of various molecular masses in cation- and anion-exchange systems, respectively. Induced salt gradients produced during these displacement experiments were measured in order to study their effect on the protein separations. The unique characteristics of these displacements were well predicted by simulations obtained from a steric mass action (SMA) ion-exchange model. These displacements differ from the traditional vision of displacement chromatography in several important ways: the isotherm of the displacer does not necessarily lie above the feed component isotherms; the concentration of the displaced proteins can sometimes exceed that of the displacer; higher-molecular-mass displacers are not necesarily more efficacious than lower-molecular-mass compounds; and the salt gradients induced by the adsorption of the displacer produce different salt micro-environments for each displaced protein.

Steric mass‐action ion exchange: Displacement profiles and induced salt gradients

AbstractThe study of nonlinear competitive equilibrium is of fundamental importance in understanding the behavior of proteins in preparative ion‐exchange chromatographic separations. In this work we present a steric mass‐action (SMA) ion‐exchange equilibrium formalism, which explicitly accounts for the steric hindrance of salt counterions upon protein binding in multicomponent equilibria. An analytical solution has been derived for the calculation of isotachic effluent profiles of displaced proteins and induced salt gradients under ideal chromatographic conditions. A stability analysis has been employed to establish the order of the feed components in the displacement train. Theoretical predictions are compared to experimental results for the separation of proteins by cation‐exchange displacement chromatography. These results demonstrate the efficacy of the SMA formalism in predicting complex behavior present in ion‐exchange displacement systems. Furthermore, the analytical solution of ideal isotachic displacement profiles with the SMA formalism enables rapid methods development and optimization of ion‐exchange displacement separations.

Cation-exchange displacement chromatography of proteins with protamine displacers: effect of induced salt gradients.

Protamine was investigated for its utility as a protein displacer in cation-exchange systems. Although the protamine solution contained several variants of the molecule, the high affinity of all of the components in this heterogeneous biopolymer enabled it to act as an efficient protein displacer. To facilitate parameter estimation of the protamine, a preliminary purification was carried out by preparative elution chromatography. Chromatographic parameters of both the feed proteins and protamine displacer were obtained for use in a multicomponent steric mass action ion-exchange displacement model. Model simulations were compared to displacement results under both moderate and intense induced salt gradient conditions. In both cases, excellent agreement was obtained between the displacement experiments and theoretical predictions. In addition, these studies serve to dramatize the importance of induced salt gradients in ion-exchange displacement systems.