Hydrophobic interaction chromatography of proteins. III. Unfolding of proteins upon adsorption

Hydrophobic interaction chromatography (HIC) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. In contrast to reversed-phase chromatography, this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. The higher the salt concentration, the lower is the amount of eluted protein. The rest can be desorbed with a buffer of low salt concentration or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochemical measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Generalizing a two-conformation model for describing salt and temperature effects on protein retention and stability in hydrophobic interaction chromatography

A two-conformation adsorption model that includes the effects of salt concentration and temperature on both stability and adsorption has been developed to describe the effects of secondary protein unfolding on hydrophobic interaction chromatography (HIC). The model has been applied to a biotech protein and to beta-lactoglobulin on Phenyl Sepharose 6FF low sub HIC media. Thermodynamic property models for adsorption and protein stability with parameters obtained from experimental chromatographic data successfully describe observed chromatographic behavior over ranges of temperature and salt concentration, provide predictions of distribution among different conformers, and give a basis for calculating trends in retention strength and stability with changing conditions, that might prove useful in HIC process development.     

Breakthrough Model of Recombinant Human-Like Collagen in Immobilized Metal Affinity Chromatography

The adsorption of recombinant human-like collagen by metal chelate media was investigated in a batch reactor and in a fixed-bed column. The adsorption equilibrium and kinetics had been studied by batch adsorption experiments. Equilibrium parameters and protein diffusivities were estimated by matching the models with the experimental data. Using the parameters of equilibrium and kinetics, various models, such as axial diffusion model, linear driving force model, and constant pattern model, were used to simulate the breakthrough curves on the columns. As a result, the most suitable isotherm was the Langmuir–Freundlich model, and the ionic strength had no effect on the adsorption capacity of chelate media. In addition, the pore diffusion model fitted very well to the kinetic data. The pore diffusivities decreased with increasing the initial protein concentration, however had little change with the ionic strength. The results also indicated that the models predict breakthrough curves reasonably well to the experimental data, especially at low initial protein concentration (0.3 mg ml⁻¹) and low flow rate (34 cm h⁻¹). By the results, we optimized the experimental conditions of a chromatographic process using immobilized metal affinity chromatography to purify recombinant human-like collagen.     

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.     

Preparation of a weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography stationary phase for protein separation using click chemistry

In this study, 3‐diethylamino‐1‐propyne was covalently bonded to the azide‐silica by a click reaction to obtain a novel dual‐function mixed‐mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high‐salt‐concentration mobile phase and weak anion exchange character in a low‐salt‐concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed‐mode chromatography stationary phase, a new off‐line two‐dimensional liquid chromatography technology using only a single dual‐function mixed‐mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.     

Comparison of the protein adsorption selectivity of salt-promoted agarose-based adsorbents: Hydrophobic, thiophilic and electron donor–acceptor adsorbents

Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol–divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene–divinyl sulfone agarose (DVS–TCP gel), tricyanoamino-propene–bisoxirane agarose (bisoxirane–TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor–acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor–acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for ‘hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor–acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor–acceptor ligand devoid of sulfur atoms.     

An Analysis of the Interactions of BSA with an Anion-Exchange Surface Under Linear and Non-Linear Conditions

The interactions of BSA with an anion-exchange adsorbent have been studied to aid in the understanding of protein adsorption in ion-exchange chromatography. Linear chromatography, flow microcalorimetry and isotherm measurements were used to analyze adsorption energetics in the linear and overloaded regions of the equilibrium isotherm. The effects of salt type, salt and protein concentration, and temperature are reported. It was observed that under all conditions studied the adsorption process was entropically driven. This was contrary to expectations, since at the pH selected ion exchange is expected to dominate. A major driving force for the adsorption of BSA on the anion exchanger was concluded to be the increase in entropy from the release of water due to interactions between hydrophobic regions on the protein and adsorbent. The data further suggest that the conformational entropy change accompanying protein adsorption on the ion exchanger may also be significant.     

Kinetics of conformational changes of fibronectin adsorbed onto model surfaces

Fibronectin (FN), a large glycoprotein found in body fluids and in the extracellular matrix, plays a key role in numerous cellular behaviours. We investigate FN adsorption onto hydrophilic bare silica and hydrophobic polystyrene (PS) surfaces using Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) in aqueous medium. Adsorption kinetics using different bulk concentrations of FN were followed for 2h and the surface density of adsorbed FN and its time-dependent conformational changes were determined. When adsorption occurs onto the hydrophilic surface, FN molecules keep their native conformation independent of the adsorption conditions, but the amount of adsorbed FN increases with time and the bulk concentration. Although the protein surface density is the same on the hydrophobic PS surface, this has a strong impact on the average conformation of the adsorbed FN layer. Indeed, interfacial hydration changes induced by adsorption onto the hydrophobic surface lead to a decrease in unhydrated beta-sheet content and cause an increase in hydrated beta-strand and hydrated random domain content of adsorbed FN. This conformational change is mainly dependent on the bulk concentration. Indeed, at low bulk concentrations, the secondary structures of adsorbed FN molecules undergo strong unfolding, allowing an extended and hydrated conformation of the protein. At high bulk concentrations, the molecular packing reduces the unfolding of the stereoregular structures of the FN molecules, preventing stronger spreading of the protein.     

Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry

Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and β‐lactoglobulin to Toyopearl Butyl‐650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases. Experiments were carried out at two different salt concentrations and five different temperatures. Protein unfolding, as indicated by large changes of molar enthalpy of adsorption Δh_ads, was observed to be dependent on temperature and salt concentration. Δh_ads were significantly higher for bovine serum albumin and ranged from 578 (288 K) to 811 (308 K) kJ/mol for 1.2 mol/kg ammonium sulfate. Δh_ads for β‐lactoglobulin ranged from 129 kJ/mol (288 K) to 186 kJ/mol (308 K). For both proteins, Δh_ads increased with increasing temperature. The influence of salt concentration on Δh_ads was also more pronounced for bovine serum albumin than for β‐lactoglobulin. The comparison of retention analysis evaluated by the van't Hoff algorithm shows that beyond adsorption other processes occur simultaneously. Further interpretation such as unfolding upon adsorption needs other in situ techniques.     

蛋白质在疏水色谱中的变性热力学

研究21-80℃温度范围内一些蛋白质和小分子在疏水相互作用色谱中的热行为。利用Van't Hoff作图(lnk'-1/T)测定蛋白质分子的热力学参数(ΔH°, ΔS°和ΔG°), 根据标准熵变(ΔS°)和标准自由能变(ΔG°)判断蛋白质在色谱过程中的构象变化, 通过ΔH°-ΔS°的线性关系估计蛋白质变性时的"补偿温度"(β), 鉴定蛋白质在疏水相互作用色谱中保留机理的同一性。     

Influence of the sample-solvent on protein retention,mass transfer and unfolding kinetics in hydrophobic interaction chromatography

Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses. In this study, these phenomena were analyzed based on the retention behavior of two model proteins, i.e., lysozyme and bovine serum albumin. Retention of these proteins was accompanied by strong band broadening originated from slow rates of mass transfer and/or of adsorption–desorption process involving the protein conformational changes. The mass transport resistances and unfolding kinetics were found to contribute to the sample-solvent effects. To avoid band deformations the process variables such as the salt concentration and temperature were adjusted in such a way that complete resolution between band profile of the sample-solvent and the protein was achieved. For the process simulation a dynamic model, which accounted for underlying kinetics was used. General guidelines of the process design were developed.     

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.     

Protein adsorption isotherm behavior in hydrophobic interaction chromatography

The adsorption behavior of proteins in hydrophobic interaction chromatography (HIC) was evaluated by determining the isotherms of a wide range of proteins on various HIC resin systems. Parallel batch experiments were carried out with eleven proteins on three hydrophobic resins with different ligand chemistries and densities. The effects of salt concentration, resin chemistry and protein properties on the isotherms were also examined. The resulting isotherms exhibited unique patterns of adsorption behaviors. For certain protein-resin combinations, a "critical salt behavior" was observed where the amount of protein bound to the resin increased significantly above this salt concentration. Proteins that exhibited this behavior tended to be relatively large with more solvent accessible hydrophobic surface area. Further, calculations indicated that under these conditions the occupied surface area of the adsorbed protein layer could exceed the accessible surface area. The establishment of unique classes of adsorption behavior may shed light on our understanding of the behavior of proteins in HIC systems.     

Recombinant protein purification using gradient-assisted simulated moving bed hydrophobic interaction chromatography. Part I: selection of chromatographic system and estimation of adsorption isotherms

The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate.     

High-throughput isotherm determination and thermodynamic modeling of protein adsorption on mixed mode adsorbents

The thermodynamic modeling of protein adsorption on mixed-mode adsorbents functionalized with ligands carrying both hydrophobic and electrostatic groups was undertaken. The developed mixed mode isotherm was fitted with protein adsorption data obtained for five different proteins on four different mixed mode adsorbents by 96-well microtitre plate high throughput batch experiments on a robotic workstation. The developed mixed mode isotherm was capable of describing the adsorption isotherms of all five proteins (having widely different molecular masses and iso-electric points) on the four mixed mode adsorbents and over a wide range of salt concentrations and solution pH, and provided a unique set of physically meaningful parameters for each resin-protein-pH combination. The model could capture the typically observed minimum in mixed mode protein adsorption and predict the precise salt concentration at which this minimum occurs. The possibility of predicting the salt concentration at which minimum protein binding occurs presents new opportunities for designing better elution strategies in mixed mode protein chromatography. Salt-protein interactions were shown to have important consequences on mixed mode protein adsorption when they occur. Finally, the mixed mode isotherm also gave very good fit with literature data of BSA adsorption on a different mixed mode adsorbent not examined in this study. Hence, the mixed mode isotherm formalism presented in this study can be used with any mixed mode adsorbent having the hydrophobic and electrostatic functional groups. It also provides the basis for detailed modeling and optimization of mixed mode chromatographic separation of proteins.     

Separation of proteins by hydrophobic interaction chromatography at low salt concentration

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.     

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.     

Mathematical model using non-uniform flow distribution for dynamic protein breakthrough with membrane adsorption media

A mathematical model has been investigated to predict protein breakthrough during membrane adsorption/chromatography operations. The new model incorporates a non-uniform boundary condition at the column inlet to help describe the deviation from plug flow within real membrane adsorption devices. The model provides estimated breakthrough profiles of a binding protein while explicitly accounting for non-uniform flow at the inlet of the separation operation by modeling the flow distribution by a polynomial. We have explored experimental breakthrough curves produced using commercial membrane adsorption devices, as well as novel adsorption media of nanolayered nanofiber membranes, and compare them to model predictions. Further, the impact of using various simplifying assumptions is considered, which can have a dramatic effect on the accuracy and predictive ability of the proposed models. The new model, using only simple batch equilibrium and kinetic uptake rate data, along with membrane properties, is able to accurately predict the non-uniform and unsymmetrical shape for protein breakthrough during operation of membrane adsorption/chromatography devices.