Modeling of adsorption in hydrophobic interaction chromatography systems using a preferential interaction quadratic isotherm

A preferential interaction quadratic isotherm model for hydrophobic interaction chromatographic systems is presented in this paper. In this isotherm, the nonlinear effect of salt on the capacity factor is described using the preferential interaction model developed by Perkins et al. [J. Chromatogr. A, 766 (1997) 1]. This is then coupled with a quadratic nonlinear isotherm to describe nonlinear adsorption behavior at high solute concentrations. The resulting preferential interaction quadratic isotherm is examined for its ability to describe solute adsorption behavior under both linear and nonlinear conditions over a wide range of salt concentrations in HIC systems. The results indicate that this isotherm is well suited for predicting nonlinear adsorption behavior in HIC systems for both proteins and low-molecular mass HIC displacers.     

Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process

Recent efforts to improve hydrophobic interaction chromatography (HIC) for use in monoclonal antibody (mAb) purification have focused on two approaches: optimization of resin pore size to facilitate mAb mass transport, and use of novel hydrophobic charge induction (HCIC) mixed mode ligands that allow capture of mAbs under low salt conditions. We evaluated standard HIC and new generation HIC and HIC-related chromatography resins for mAb purification process efficiency and product quality both as isolated chromatography steps and in purification process trains. We find that HIC resins with optimized pore size have significantly improved binding capacity which can increase HIC purification unit operation efficiency. The HCIC Mercapto-Ethyl-Pyridine (MEP) resin, which shows a different salt impact trend and impurity resolution pattern from standard HIC resin, can not only capture mAb from crude CHO fermentation supernatant but also substantially enhance mAb purification process flow efficiency when serving as a polishing role.     

High-throughput protein precipitation and hydrophobic interaction chromatography: salt effects and thermodynamic interrelation

Salt-induced protein precipitation and hydrophobic interaction chromatography (HIC) are two widely used methods for protein purification. In this study, salt effects in protein precipitation and HIC were investigated for a broad combination of proteins, salts and HIC resins. Interrelation between the critical thermodynamic salting out parameters in both techniques was equally investigated. Protein precipitation data were obtained by a high-throughput technique employing 96-well microtitre plates and robotic liquid handling technology. For the same protein-salt combinations, isocratic HIC experiments were performed using two or three different commercially available stationary phases-Phenyl Sepharose low sub, Butyl Sepharose and Resource Phenyl. In general, similar salt effects and deviations from the lyotropic series were observed in both separation methods, for example, the reverse Hofmeister effect reported for lysozyme below its isoelectric point and at low salt concentrations. The salting out constant could be expressed in terms of the preferential interaction parameter in protein precipitation, showing that the former is, in effect, the net result of preferential interaction of a protein with water molecules and salt ions in its vicinity. However, no general quantitative interrelation was found between salting out parameters or the number of released water molecules in protein precipitation and HIC. In other words, protein solubility and HIC retention factor could not be quantitatively interrelated, although for some proteins, regular trends were observed across the different resins and salt types.     

Effect of salts and temperature on the adsorption of bovine serum albumin on polypropylene glycol-Sepharose under linear and overloaded chromatographic conditions

The interaction thermodynamics associated with bovine serum albumin (BSA) adsorption on polypropylene glycol (PPG)-Sepharose CL-6B gel, using ammonium and sodium sulfate was studied. Analysis of data under linear conditions was accomplished with the stoichiometric displacement retention model and preferential interaction approach. Preferential interaction analysis indicated a strong entropic driving force due to the release of a large amount of solvent on adsorption. Flow microcalorimetry provided direct heat of adsorption measurements under overloaded conditions and confirmed that the adsorption of BSA on PPG-Sepharose was entropically driven within the range of conditions studied. Using these data in combination with isotherm measurements, it is shown that protein surface coverage, salt concentration, salt type and temperature affect the enthalpic and entropic behavior in hydrophobic interaction chromatography (HIC). This study shows that protein-sorbent interactions can be strongly influenced by the degree of water release, protein-protein interactions on the surface, and the re-orientation and/or reconfiguration of the adsorbed protein.     

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

A novel dual‐retention mechanism mixed‐mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high‐salt‐concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low‐salt‐concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion‐exchange chromatography and HIC columns. The results indicated that the novel dual‐retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a ‘2D column’. In addition, the mixed retention mechanism of proteins on this ‘2D column’ was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual‐retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this ‘2D column’, a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd.     

A new thermodynamic model describes the effects of ligand density and type,salt concentration and protein species in hydrophobic interaction chromatography

A new thermodynamic model is derived that describes both loading and pulse-response behavior of proteins in hydrophobic interaction chromatography (HIC). The model describes adsorption in terms of protein and solvent activities, and water displacement from hydrophobic interfaces, and distinguishes contributions from ligand density, ligand type and protein species. Experimental isocratic response and loading data for a set of globular proteins on Sepharose™ resins of various ligand types and densities are described by the model with a limited number of parameters. The model is explicit in ligand density and may provide insight into the sensitivity of protein retention to ligand density in HIC as well as the limited reproducibility of HIC data.     

Adsorption affinity of certain biomolecules onto polymeric resins: Interpretation from molecular orbital theory

Molecular interactions have been studied for adsorption of certain biomolecules in aqueous solutions using two different types of polymeric resins as adsorbents. Molecular modeling study is based on molecular orbital theory. Adsorption affinity expresses as the slope of the linear region of the isotherm for a solute is found to be different for different adsorbents, and this difference can be interpreted from the differences in sorbent surface chemistry and morphological structure. The adsorptive interaction on the polymeric resins computed on the basis of frontier orbital theory seems to correlate well with the experimentally measured adsorption affinity. Electronic states of adsorbent and adsorbate were calculated using the semiempirical molecular orbital (MO) method from which energy of adsorption in aqueous solution was estimated. It was found that charge transfer interaction plays an important role in the adsorption of certain biomolecules on aqueous solution. The experimentally measured enthalpy of adsorption seems to correlate well with the adsorptive interaction energy computed from molecular orbital theory.     

A novel thermodynamic state recursion method for description of nonideal nonlinear chromatographic process of frontal analysis

A novel thermodynamic state recursion (TSR) method, which is based on nonequilibrium thermodynamic path described by the Lagrangian-Eulerian representation, is presented to simulate the whole chromatographic process of frontal analysis using the spatial distribution of solute bands in time series like as a series of images. TSR differs from the current numerical methods using the partial differential equations in Eulerian representation. The novel method is used to simulate the nonideal, nonlinear hydrophobic interaction chromatography (HIC) processes of lysozyme and myoglobin under the discrete complex boundary conditions. The results show that the simulated breakthrough curves agree well with the experimental ones. The apparent diffusion coefficient and the Langmuir isotherm parameters of the two proteins in HIC are obtained by the state recursion inverse method. Due to its the time domain and Markov characteristics, TSR is applicable to the design and online control of the nonlinear multicolumn chromatographic systems.     

Effect of surface hydrophobicity distribution on retention of ribonucleases in hydrophobic interaction chromatography

The effect of surface hydrophobicity distribution of proteins on retention in hydrophobic interaction chromatography (HIC) was investigated. Average surface hydrophobicity as well as hydrophobic contact area between protein and matrix were estimated using a classical thermodynamic model. The applicability of the model to predict protein retention in HIC was investigated on ribonucleases with similar average surface hydrophobicity but different surface hydrophobicity distribution. It was shown experimentally that surface hydrophobicity distribution could have an important effect on protein retention in HIC. The parameter "hydrophobic contact area," which comes from the thermodynamic model, was able to represent well the protein retention in HIC with salt gradient elution. Location and size of the hydrophobic patches can therefore have an important effect on protein retention in HIC, and the hydrophobic contact area adequately describes this.     

Dynamic control of protein conformation transition in chromatographic separation based on hydrophobic interactions: Molecular dynamics simulation

Conformational transitions of a protein in hydrophobic interaction based chromatography, including hydrophobic interaction chromatography (HIC) and reversed-phase liquid chromatography (RPLC), and their impact on the separation process and performance were probed by molecular dynamics simulation of a 46-bead β-barrel coarse-grained model protein in a confined pore, which represents the porous adsorbent. The transition of the adsorbed protein from the native conformation to an unfolded one occurred as a result of strong hydrophobic interactions with the pore surface, which reduced the formation of protein aggregates. The conformational transition was also displayed in the simulation once an elution buffer characterized by weaker hydrophobicity was introduced to strip protein from pore surface. The discharged proteins that underwent conformational transition were prone to aggregation; thus, an unsatisfactory yield of the native protein was obtained. An orthogonal experiment revealed that in addition to the strengths of the protein–protein and protein–adsorbent hydrophobic interactions, the elution time required to reduce the above-mentioned interactions also determined the yield of native protein by HIC and RPLC. Stepwise elution, characterized by sequential reduction of the hydrophobic interactions between the protein and adsorbent, was presented as a dynamic strategy for tuning conformational transitions to favor the native conformation and reduce the formation of protein aggregates during the elution process. The yield of the native protein obtained by this dynamic operation strategy was higher than that obtained by steady-state elution. The simulation study qualitatively reproduced the experimental observations and provided molecular insight that would be helpful for designing and optimizing HIC and RPLC separation of proteins.     

疏水相互作用色谱中Z值的测定

对在疏水相互作用色谱中报道的３种测定蛋白质Ｚ值的方法进行了讨论和比较，从线性参数，各种Ｚ值的物理意义，Ｚ值的准确性等结果看，计量置换保模型中的计量参数Ｚ是一个在ＨＩＣ中好的和通用的描述蛋白保留特征的表征参数。     

Concentration-dependent self-diffusion of liquids in nanopores: a nuclear magnetic resonance study

Nuclear magnetic resonance has been applied to study the details of molecular motion of low-molecular-weight polar and nonpolar organic liquids in nanoporous silicon crystals of straight cylindrical pore morphology at different pore loadings. Effective self-diffusion coefficients as obtained using the pulsed field gradient nuclear magnetic resonance method were found to pass through a maximum with increasing concentration for all liquids under study. Taking account of a concentration-dependent coexistence of capillary condensed, adsorbed and gaseous phases a generalized model for the effective self-diffusion coefficient was developed and shown to satisfactorily explain the experimental results. An explicit use of the adsorption isotherm properties within the model extends its applicability to the mesoporous range and highlights the role of surface interaction for the transport of molecules in small pores. The problem of surface diffusion and diffusion of multilayered molecules is also addressed.     

Retention Behavior of Polyethylene Glycol and Its Influence on Protein Elution on Hydrophobic Interaction Chromatography Media

The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface. The effect enhanced with increasing molecular weight of the polymer and hydrophobicity of the HIC media. Addition of PEG to the mobile phase reduced binding of proteins to HIC resins, which was demonstrated with two model systems: lysozyme (LYZ) and immunoglobulin G (IgG), and their mixtures. In case of LYZ, the presence of PEG caused reduction in the protein retention, whereas for IgG—a decrease in efficiency of the protein capture. The effect depended on the adsorption pattern of PEG; it was pronounced in the systems in which conformational changes of the polymer were suggested to occur.     

A parallel pore and surface diffusion model for predicting the adsorption and elution profiles of lispro insulin and two impurities in gradient-elution reversed phase chromatography

Lispro insulin (LPI), a widely used insulin analog, is produced on tons per year scale. Linear gradient reversed phase chromatography (RPC) is used in the production to separate LPI from two impurities, which differ from LPI by a single amino acid residue. A chromatography model for the ternary separation in this RPC process is unavailable from the literature. In this study, a parallel pore and surface diffusion model is developed and verified for LPI and the two impurities. The LPI can be recovered with high yield (≥95%) and high purity (>99.5%). A new method, which requires a small amount of materials and an order of magnitude fewer experiments, has been developed to estimate the solvent-modulated isotherm parameters. A modified reversed phase modulator model is developed to correlate the adsorption isotherms of LPI and impurities. A strategy has been developed for estimating the intrinsic pore diffusivity and surface diffusivity. Since the adsorption affinities decrease by more than three orders of magnitude as organic fraction (φ) increases from 0.19 to 0.40, the apparent diffusivities based on a pore diffusion model or a surface diffusion model can also vary by several orders of magnitude. For this reason, a pore diffusion model or a surface diffusion model with a constant apparent diffusivity cannot predict closely the chromatograms over the same range of organic fractions, concentrations, and loadings. The parallel pore and surface diffusion model with constant diffusivities can predict closely the frontal and elution profiles over a wide range of organic fractions (0.19-0.40), LPI concentrations (0.05-18 g/L), linear velocities (<10 cm/min), and loading volume (0.0004-13 CV). For large loading stepwise and linear gradient elution, the peaks of LPI and the impurities are strongly focused by self-sharpening and gradient focusing effects as a result of the steep decrease of adsorption affinity from the loading φ (0.19) to elution φ (≥0.27). When the ratio of diffusion rate to convection rate is greater than 10, spreading due to diffusion is largely compensated by the focusing effects. As a result, a pore diffusion model with a constant pore diffusivity can predict closely the elution profiles in stepwise and linear gradient elution. The experimental yield values (≥95%) can be predicted to within ±1% by the model.     

Contribution of concentration-dependent surface diffusion in ternary adsorption kinetics of ethane, propane and n-butane in activated carbon

The role of concentration-dependent surface diffusion in the adsorption kinetics of a multicomponent system is investigated in this paper. Ethane, propane and n-butane are selected as the model adsorbates and Ajax activated carbon as the model adsorbent. Adsorption equilibrium isotherm and dynamic parameters extracted from single-component systems are used to predict the ternary adsorption equilibria and kinetics. The effect of concentration-dependent surface diffusion on the adsorption kinetics predictions is studied by comparing the results of two mathematical models with the experimental data. Three diffusion mechanisms, macropore, surface and micropore diffusions are incorporated in both models. The distinction between these two models is the use of the chemical potential gradient as the driving force for the diffusion of the adsorbed species in one model and the concentration gradient in the other. It was found that the model using the chemical potential gradient provides a better prediction of the ternary adsorption kinetics data, suggesting the importance of the concentration dependency of the surface diffusion, which is implicitly reflected in the chemical potential gradient. The kinetic model predictions are also affected by the way how single-component adsorption equilibrium isotherm data are fitted.     

The effects of ligand chain length, salt concentration and temperature on the adsorption of bovine serum albumin onto polypropyleneglycol-Sepharose

The interaction thermodynamics associated with bovine serum albumin adsorption on polypropylene glycol (n=3)-Sepharose CL-6B and polypropylene glycol (n=7)-Sepharose CL-6B, using ammonium sulfate as the modulator was studied. Analysis of data under linear conditions was accomplished with the stoichiometric displacement retention model, preferential interaction approach and van't Hoff plots applied to HIC systems. Preferential interaction analysis indicated a strong entropic driving force under linear conditions, due to the release of a large amount of solvent on adsorption. In contrast, flow microcalorimetry under overloaded conditions showed that the adsorption of bovine serum albumin may be entropically or enthalpically driven. It is postulated that adsorption in the nonlinear region is influenced by the degree of water release, protein-protein interactions on the surface, reorientation of ligand, and conformational changes in the protein.     

Purification of antibody heteropolymers using hydrophobic interaction chromatography

A heteropolymer (HP) is a unique dual antibody conjugate composed of specific, chemically cross-linked monoclonal antibodies (mAbs). In this study we have demonstrated that HPs can be purified using hydrophobic interaction chromatography (HIC). Two propyl HIC resins; [PolyPropyl A and EMD Fractogel Propyl (S)] were evaluated in this study. Phosphate buffers, pH 6.5 containing ammonium sulfate or sodium sulfate were used to bind the HP to the column. A descending sulfate gradient or step gradient was used to elute the bound HP species from the column. The HP reaction mixture typically contains multiple conjugated HP species, as well as unreacted monomer mAbs. Conjugated HP product was successfully separated from unreacted antibody monomers with both propyl resins using buffers with ammonium sulfate. There was no monomer separation from HP using buffers with sodium sulfate. The purification processes, presented in this study allows the non-cross-linked antibodies to pass through the column without being bound to the resin, while the cross-linked antibodies (the HP product) bound to the column were subsequently eluted by decreasing the ammonium sulfate concentration in the running buffer. HP product was efficiently separated from free mAbs using Propyl HIC resins at both analytical and preparative scales.     

Analysis of diffusion models for protein adsorption to porous anion-exchange adsorbent

The ion-exchange adsorption kinetics of bovine serum albumin (BSA) and gamma-globulin to an anion exchanger, DEAE Spherodex M, has been studied by batch adsorption experiments. Various diffusion models, that is, pore diffusion, surface diffusion, homogeneous diffusion and parallel diffusion models, are analyzed for their suitabilities to depict the adsorption kinetics. Protein diffusivities are estimated by matching the models with the experimental data. The dependence of the diffusivities on initial protein concentration is observed and discussed. The adsorption isotherm of BSA is nearly rectangular, so there is little surface diffusion. As a result, the surface and homogeneous diffusion models do not fit to the kinetic data of BSA adsorption. The adsorption isotherm of gamma-globulin is less favorable, and the surface diffusion contributes greatly to the mass transport. Consequently, both the surface and homogeneous diffusion models fit to the kinetic data of gamma-globulin well. The adsorption kinetics of BSA and gamma-globulin can be very well fitted by parallel diffusion model, because the model reflects correctly the intraparticle mass transfer mechanism. In addition, for both the favorably bound proteins, the pore diffusion model fits the adsorption kinetics reasonably well. The results here indicate that the pore diffusion model can be used as a good approximate to depict protein adsorption kinetics for protein adsorption systems from rectangular to linear isotherms.     

Separation of Proteins on Polar Bonded Phases by Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) with a polar bonded phase (“Acetamide”) developped for size exclusion chromatography (SEC) is described. Retention of proteins depends on the surface area of the stationary phase, the pH and ionic strength of the eluent. For efficient separation the pore diameter should be 25 nm or more. The surface area should be large to achieve retention even at low ionic strength. Separation is only possible with a gradient from high to low ionic strength. Gradient volumes of 10 empty column volumes with column lengths above 15 cm are recommended. Selectivity can be optimized via pH adjustment. The advantage of this column packing is its applicability for two different separation modes: SEC and HIC.     

Uptake of alkanes and alcohols by ion-exchange resins in aqueous solution.

Uptake of alkane (C5-C9) and alcohol (C4-C7) solutes by both strong acid cation exchange resins and strong base anion exchange resins in aqueous medium has been studied. The amount of solute taken up by resins is directly proportional to the solute concentration equilibrium with resins. Hydrophobic interaction between the solute and the resin matrix appears to play an important role in the uptake phenomenon.