Steric Mass Action Model Research Articles

Modeling and robust pooling design of a preparative cation-exchange chromatography step for purification of monoclonal antibody monomer from aggregates

This study has implemented and calibrated a model that describes the separation of the monomer of monoclonal antibodies from the dimer and larger oligomers on preparative-scale using cation-exchange chromatography. A general rate model with temperature dependent diffusion was coupled to a pH- and temperature-dependent steric mass action model. The model was shown to predict the retention of the monomer, dimer, and oligomer at low loadings for different pH levels and temperatures. Additionally, the model was shown to adequately predict the elution behavior of the monomer and soluble aggregates at high loadings within the same ranges with some limitations. The model was not able to accurately describe the shape of the product break-through curves or the slight levels of co-elution of the dimer and oligomer with the monomer at higher pH. The model was used to predict how 12 process variations impact the separation. The model is used to establish an elution end collection criterion such that the step can robustly provide the target purity of monomers.

Model‐based process development for the purification of a modified human growth hormone using multimodal chromatography

This study demonstrates how the multimodal Capto adhere resin can be used in concert with calcium chloride or arginine hydrochloride as mobile phase modifiers to create a highly selective purification process for a modified human growth hormone. Importantly, these processes are shown to result in significant clearance of product related aggregates and host cell proteins. Furthermore, the steric mass action model is shown to be capable of accurately describing the chromatographic process and the aggregate removal. Finally, justification of the selected operating ranges is evaluated using the model together with Latin hypercube sampling. The results in this article establish the utility of multimodal chromatography when used with appropriate mobile phase modifiers for the downstream bioprocessing of a modified human growth hormone and offer new approaches for bioprocess verification.

Thermodynamic description of peptide adsorption on mixed-mode resins

In this work the adsorption of tri-peptides on a mixed-mode resin was studied using isocratic pulse response experiments. Various salt concentration, temperature and pH combinations were used to measure retention times of several tri-peptides. The experiments were evaluated according to an extension of the stoichiometric displacement model and the steric mass action model of protein–ligand binding. The application of this model in the understanding of mixed mode adsorption process is discussed. A unique set of meaningful thermodynamic parameters was obtained for each resin-peptide-temperature and resin-peptide-pH combination. Finally it was shown that these thermodynamic parameters can be used in defining quantitative relationships within the framework of extra thermodynamic relationships.

Methods development in multimodal chromatography with mobile phase modifiers using the steric mass action model

The ability to predict downstream protein purification processes is of great value in the biopharmaceutical industry; saving time, cost and resources. While many complex models exist, the appropriate use of simple models can be a useful tool for rapidly designing and optimizing processes as well as for risk analysis and establishing parameter ranges. In this study, the steric mass action isotherm is successfully employed to predict the chromatographic behavior of a multimodal anionic Capto adhere system in the presence of various mobile phase modifiers. An experimental protocol consisting of only a few column experiments is shown to be sufficient to establish the model. Proof of concept is carried out using human insulin and bovine serum albumin which have varying degrees of hydrophobicity, charge and size. Finally, the model predictions are verified under various experimental conditions and the unique selectivity of this multimodal system is explored and compared with traditional anion exchange resins. The simple model approach described here represents a rapid and useful method for model based process development of multimodal chromatography.

A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography

The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC.

Improving the Operating Conditions of Gradient Ion-Exchange Simulated Moving Bed for Protein Separation

Chromatographic separation processes such as simulated moving bed (SMB) are widely used in the petrochemical, fine chemical, sugar, and pharmaceutical industries. Their separation efficiency can be improved by optimizing the components’ adsorptivity in the different unit sections through, e.g., the use of solvent, temperature, or PH gradients. In this work, the salt gradient ion-exchange SMB used to separate two proteins, β-lactoglobulins A and B, is theoretically analyzed, where the protein adsorption is described by the steric mass action model. Detailed model-based sequential optimization studies have been carried out for both closed-loop isocratic SMB as well as closed and open-loop gradient ion-exchange SMB. The separation efficiency is described by the throughput. Different gradient cases were analyzed to find out the influence factors. The results show that the gradient SMB is more efficient than the isocratic SMB in terms of startup time, throughput, and desorbent-to-throughput ratio. Moreover, it can be known that using open-loop gradient ion-exchange SMB to separate proteins is more effective than using closed-loop gradient SMB. The influencing factors such as the mass transfer efficiency and the maximum flow rate on separation efficiency are discussed according to different cases.

Adsorption of Proteins onto Ion-Exchange Chromatographic Media: A Molecular Dynamics Study

Molecular dynamics (MD) simulations with coarse-grained models are used to investigate the adsorption process of proteins onto an ion-exchange chromatographic medium. The adsorption of human serum albumin (HSA) and bovine hemoglobin (bHb) on the anion exchanger Q Sepharose FF is studied. These two proteins have similar molecule sizes with different isoelectric points. To obtain a reliable set of data, simulations with different initial conditions are carried out for each protein. Convincingly, the results of the MD simulations are qualitatively consistent with those of previous experiments. HSA reaches a stable adsorption in all simulations, while the binding sites can differ. In contrast, bHb reaches a stable adsorption only in one simulation. Due to the stronger protein–ligand interaction of HSA, it adsorbs faster, stronger, and with more binding sites onto Q Sepharose FF than bHb. The spatial movements during the adsorption process and the adsorbed states are investigated in detail. Moreover, we studied the underlying physical relevance of two parameters (characteristic charge and steric factor) of the steric mass action (SMA) model for HSA and for bHb with MD simulations and compared them with the results of experiments. The results reveal that MD simulations are useful to interpret the physical consistence of the SMA parameters.

Single and binary adsorption of proteins on ion‐exchange adsorbent: The effectiveness of isothermal models

Simultaneous and sequential adsorption equilibria of single and binary adsorption of bovine serum albumin and bovine hemoglobin on Q Sepharose FF were investigated in different buffer constituents and initial conditions. The results in simultaneous adsorption showed that both proteins underwent competitive adsorption onto the adsorbent following greatly by protein-surface interaction. Preferentially adsorbed albumin complied with the universal rule of ion-exchange adsorption whereas buffer had no marked influence on hemoglobin adsorption. Moreover, an increase in initial ratios of proteins was benefit to a growth of adsorption density. In sequential adsorption, hemoglobin had the same adsorption densities as single-component adsorption. It was attributed to the displacement of preadsorbed albumin and multiple layer adsorption of hemoglobin. Three isothermal models (i.e. extended Langmuir, steric mass-action, and statistical thermodynamic (ST) models) were introduced to describe the ion-exchange adsorption of albumin and hemoglobin mixtures. The results suggested that extended Langmuir model gave the lowest deviation in describing preferential adsorption of albumin at a given salt concentration while steric mass-action model could very well describe the salt effect in albumin adsorption. For weaker adsorbed hemoglobin, ST model was the preferred choice. In concert with breakthrough data, the research further revealed the complexity in ion-exchange adsorption of proteins.

Ion exchange membrane adsorption of bovine serum albumin—Impact of operating and buffer conditions on breakthrough curves

Abstract The relevant phenomena underlying the adsorption of proteins on ion exchange membranes with grafted polymer layers are not well understood yet. In this work the influence of operating and buffer conditions on the adsorption of bovine serum albumin (BSA) on anion and cation exchange membrane adsorbers is systematically investigated in order to find indications on the relevant mechanisms. The experimental results obtained at different buffer conditions are analyzed using the steric mass action model. Large differences were observed between the adsorption behavior of BSA on anion and cation exchange membranes. On the anion exchange membranes the dynamic binding capacity at 10% breakthrough was independent of the applied flow rate and decreased with increasing salt concentration. This in contrast to the cation exchange membranes, for which the dynamic binding capacity was strongly dependent on the flow rate and for which an optimum in binding capacity was observed at an intermediate salt concentration. Especially, the results obtained on cation exchange suggest a strong role of non-ideal effects such as steric hindrance and electrostatic repulsion in membrane adsorption. An improved understanding of these effects is required for further optimization of membrane adsorber materials.

Determination of parameters for the steric mass action model—A comparison between two approaches

The application of mechanistic modeling for the optimization of chromatographic steps increased recently due to time efficiency of algorithms and rising calculation power. In the modeling of ion exchange chromatography steps, the sorption processes occurring on adsorbent particle surfaces can be simulated with the steric mass action (SMA) model introduced by Brooks and Cramer (1992) [14]. In this paper, two approaches for the determination of SMA parameters will be carried out and discussed concerning their specific experimental effort, quality of results, method differences, reasons for uncertainties and consequences for SMA parameter determination: Approach I: estimation of SMA parameters based on gradient and frontal experiments according to instructions in Brooks and Cramer (1992) [14] and Shukla et al. (1998) [16]. Approach II: application of an inverse method for parameter estimation, resulting in SMA parameters that induce a best fit of chromatographic data to a mechanistic model for column chromatography. These approaches for SMA parameter determination were carried out for three proteins (ribonuclease A, cytochrome c and lysozyme) at pH 5 and pH 7. The results were comparable and the order of parameter values and their relations to the chromatographic data similar. Nevertheless, differences in the complexity and effort of methods as well as the parameter values themselves were observed. The comparison of methods demonstrated that discrepancies depend mainly on model sensitivities and additional parameters influencing the calculations. However, the discrepancies do not affect predictivity; predictivity is high in both approaches. The approach based on an inverse method and the mechanistic model has the advantage that not only retention times but also complete elution profiles can be predicted. Thus, the inverse method based on a mechanistic model for column chromatography is the most comfortable way to establish highly predictive SMA parameters lending themselves for the optimization of chromatography steps and process control.

Impact of ligand density on the optimization of ion‐exchange membrane chromatography for viral vector purification

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development.

Adsorption of deamidated antibody variants on macroporous and dextran-grafted cation exchangers: I. Adsorption equilibrium

Single and multicomponent adsorption isotherms were obtained for deamidated variants of a monoclonal antibody on two cation exchangers with different pore structures, one with a macroporous architecture – UNOsphere S, and the other with charged dextran grafts – Capto S. No selectivity between the different deamidated forms was seen at pH 5 for either stationary phase. However, although the binding strength was lower, both media exhibited substantial selectivity at pH 7.5. The effective binding charge, determined from linear gradient elution experiments and from the steric mass action model, was different for the two different media, but remained nearly the same for the different variants suggesting that the selectivity is determined by the strength of binding, rather than by the binding charge. This result agrees with the higher binding constants determined for the less deamidated forms. At low ionic strength, the binding capacity of the dextran grafted media was much higher than that of the macroporous matrix at either pH. However, similar capacities were obtained for the two stationary phases at ∼140 mM Na + for pH 5 and at 50 mM Na + for pH 7.5. For both materials, predictions of multicomponent adsorption based on the steric mass action model were in good agreement with experimental results indicating that the different variants bind in competition with each other. In general, this work demonstrates the utility of modeling adsorption equilibrium allowing an accurate description of competitive binding, which is a necessary step for a complete description of a chromatographic separation process.

Apolipoprotein A‐IMilano anion exchange chromatography: Self association and adsorption equilibrium

The self-associative properties of apolipoprotein A-I(Milano) (apoA-I(M)) were investigated in relationship to its anion exchange behavior on Q-Sepharose-HP with and without the addition of urea as a denaturant. Self-association was dependent on protein and urea concentration and both influenced interactions of the protein with the chromatographic surface. In the absence of urea, apoA-I(M) was highly associated and existed primarily as a mixture of homodimer, tetramer and hexamer forms. Under these conditions, since the binding strength was greater for the oligomer forms, broad, asymmetrical peaks were obtained in both isocratic and gradient elution. Adding urea depressed self-association and caused unfolding. This resulted in sharper peaks but also decreased the binding strength. Thus, under these conditions chromatographic elution occurred at lower salt concentrations. The adsorption isotherms obtained at high protein loadings were also influenced by self-association and by the varying binding strength of the differently associated and unfolded forms. The isotherms were thus dependent on protein, urea, and salt concentration. Maximum binding capacity was obtained in the absence of urea, where adsorption of oligomers was shown to be dominant. Adding urea reduced the apparent binding capacity and weakened the apparent binding strength. A steric mass action model accounting for competitive binding of the multiple associated forms was used to successfully describe the equilibrium binding behavior using parameters determined from isocratic elution and isotherm experiments.

Regulation of protein multipoint adsorption on ion-exchange adsorbent and its application to the purification of macromolecules

Ion-exchange chromatography (IEC) using commercial ionic absorbents is a widely used technique for protein purification. Protein adsorption onto ion-exchange adsorbents often involves a multipoint adsorption. In IEC of multimeric proteins or “soft” proteins, the intense multipoint binding would make the further desorption difficult, even lead to the destruction of protein structure and the loss of its biological activity. In this paper, DEAE Sepharose FF adsorbents with controllable ligand densities from 0.020 to 0.183 mmol/ml were synthesized, and then the effect of ligand density on the static ion-exchange adsorption of bovine serum albumin (BSA) onto DEAE Sepharose FF was studied by batch adsorption technique. Steric mass-action (SMA) model was employed to analyze the static adsorption behavior. The results showed that the SMA model parameters, equilibrium constant ( K a), characteristic number of binding sites ( υ) and steric factor ( σ), increased gradually with ligand density. Thus, it was feasible to regulate BSA multipoint adsorption by modulating the ligand density of ion-exchange adsorbent. Furthermore, IEC of hepatitis B surface antigen (HBsAg) using DEAE Sepharose FF adsorbents with different ligand densities was carried out, and the activity recovery of HBsAg was improved from 42% to 67% when the ligand density was decreased from 0.183 to 0.020 mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041 mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins.

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.

Influence of pH and Ionic Strength on the Steric Mass-Action Model Parameters around the Isoelectric Point of Protein

The ion-exchange equilibrium and the dependence of the parameters in the steric mass-action (SMA) model on salt concentration and buffer pH around the isoelectric point of protein were studied. Bovine serum albumin (BSA, isoelectric point = 5.4) was used as a model protein and DEAE Sepharose FF as an ion exchanger. Finite batch adsorption experiments and isocratic elution chromatography were performed for the determination of the model parameters (i.e., characteristic charge, equilibrium constant, and steric factor). The results showed that pH had significant effects on the parameters. With an increase of pH from 4.5 to 6.5, the characteristic charge increased from 0.9 to 3.0 and leveled off as a plateau at pH above 5.5. The charge groups in the contact region of protein surface were considered to play a crucial role on the characteristic charge. The decrease of pH and increase of salt concentration lowered the absolute value of the zeta potential of the protein surface and led to a decrease of the equilibrium constant. The steric factor remained unchanged at about 31 at pH 5.5 and 6.0 and increased to 44.5 at pH 5.0 and 96.8 at pH 4.5, mainly as a result of the lower adsorption capacity of BSA at pH <5.5. Furthermore, the increase of the molecular volume of BSA at pH 4.5 would be an additional reason for the increase of the steric factor. Taking into account the effect of the pH and salt concentration on these parameters, the SMA model described the ion exchange equilibrium of protein more accurately.

Chemically Selective Displacers for High-Resolution Protein Separations in Ion-Exchange Systems: Effect of Displacer−Protein Interactions

Displacer lead compounds were selected from a commercially available database to identify potential selective displacers for a binary protein mixture in ion exchange chromatography. Parallel batch screening experiments were carried out with these lead compounds to study the effect of displacer concentration on the relative amounts of the proteins displaced. Experiments were conducted with a mixture containing ribonuclease A and alpha-chymo-trypsinogen A which exhibited very similar retention behavior under linear gradient conditions. The batch displacement results indicated that most of these lead compounds were indeed selective for displacing ribonuclease A. In fact, one of these displacers exhibited extremely high selectivity, displacing essentially all of the ribonuclease A while displacing minimal alpha-chymotrypsinogen A at a displacer concentration of 10 mM. These results were validated under column conditions, with the ribonuclease A being displaced and the alpha-chymotrypsinogen A remaining on the column after the displacer breakthrough. In order to examine whether this was mass action or chemically selective displacement, an affinity ranking plot based on the Steric Mass Action (SMA) model was generated, and the results confirmed that this was not a mass action displacement. In order to test the hypothesis that displacer protein binding was playing a role in these separations, Surface Plasmon Resonance (SPR) was carried out. The results suggest that while the chemically selective displacer interacted with alpha-chymo-trypsinogen A, it had no interaction with ribonuclease A. The ability to exploit protein displacer binding in concert with appropriate displacer resin affinities opens up new possibilities for creating selective displacement systems.

Anion-exchange membrane chromatography for purification of rotavirus-like particles

Anion-exchange chromatography is one of the separation processes of choice for the recovery/purification of proteins and complex bio-structures as viruses or virus-like particles (VLPs), e.g., rotavirus-like particles (RLPs) representing a potential biopharmaceutical, where a safer vaccine technology is endeavored. Purification strategies of complex bioparticles are as yet rather empirical. The breakthrough pursued hereby was to gain theoretical and predictive knowledge on the anion-exchange membrane chromatography processing of this type of products. Currently, the use of membrane chromatography is expanding due to its scalability, robustness and disposable nature, allowing high fluxes, rapid processing, little buffer consumption and a validation-free environment. A novel matrix, Sartobind™ D membrane adsorber from Sartorius, was used and thoroughly studied for the adsorption of VLPs. Screening studies showed the sensitive effect of the ionic strength and the pH of the equilibration buffer on product final recovery. Steric mass action (SMA) model formulation was implemented for the prediction of the elution profile of purified RLP pulses. Experimental data fitting gave good model validation therefore yielding insights on the fundamental understanding of the ion-exchange separation mechanism of virus-like particles and, potentially, for other similar structures. Anion-exchange membrane chromatography was finally used in a larger scale downstream process, confirming that rotavirus VLPs can be reproductively purified to clinical grade at 46% global recovery yield, concomitant with nearly 100% removal of host bulk DNA and approximately 98% of host cell proteins.

Simulation of a human serum albumin downstream process incorporating ion-exchange membrane adsorbers

In this paper a generic process model for the simulation of various downstream processes for protein purification is presented. It consists of detailed, dynamic models for ion-exchange membrane adsorbers, ion-exchange and size-exclusion chromatography and ultra-/diafiltration. For the implemented ion-exchange membrane adsorber model, the steric mass action model has been applied. The isotherm parameters have been determined experimentally for bovine serum albumin (BSA) and immunoglobulin G (IgG). The comparison between simulation results of the ion-exchange membrane and experiments for two protein mixtures containing BSA and IgG shows satisfactory agreement. Two case studies based on the purification process of human serum albumin are performed to illustrate the influence of structural changes in the process flowsheet on the process performance. The simulation results show that the replacement of an anion-exchange chromatography column by an anion-exchange membrane adsorber leads to a significant reduction in overall process time at constant yield and purity. The case studies demonstrate the applicability of the generic process model for detailed process analysis.

Adsorption equilibrium of fructosyltransferase on a weak anion-exchange resin

The adsorption equilibrium of a glycoprotein, fructosyltransferase from Aureobasidium pullulans, on an anion-exchange resin, Sepabeads FP-DA activated with 0.1 M NaOH, was investigated. The adsorption isotherms were determined at 20 °C in a phosphate–citrate buffer with pH 6.0 using the static method. Sodium chloride was used to adjust the ionic strength in the range from 0.0215 to 0.1215 mol dm −3 which provided conditions varying from a weak effect of salt concentration on protein binding to its strong suppression. The equilibrium data were very well fitted by means of the steric mass-action model when the ion-exchange capacity of 290 mmol dm −3 was obtained from independent frontal column experiments. The model fit provided the protein characteristic charge equal to 1.9, equilibrium constant 0.326, and steric factor 1.095 × 10 5.