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.     

Multiple-injection technique for isolating a target protein from multicomponent mixtures

An integrated chromatographic process comprising ion exchange (IEC) and hydrophobic interaction chromatography (HIC) for isolating a target protein form multicomponent mixtures has been analyzed. The model mixture contained immunoglobulin G that was the key product of the separation process, cytochrome C and ovalbumin. The adsorption characteristics and the mass transport kinetics of the model proteins have been determined along with their dependencies on the operating variables such as pH, temperature and the salt concentration for IEC as well as HIC media. Limitations of the process efficiency resulting from kinetic effects, solubility constraints and the necessity of the mobile phase exchange between chromatographic steps have been discussed. To improve the performance of the integrated process the multiple-injection technique has been suggested. This technique consisted in loading feed mixtures dissolved in a good solvent onto the column by several small-volume injections under conditions of strong protein adsorption. It allowed diminishing interactions between the sample-solvent and protein and elimination of undesired effects such as band splitting and band broadening. For the process design and optimization a dynamic model has been used accounting for thermodynamics and kinetics of the process. The optimization results indicated superiority of the multiple-injection technique over standard isocratic injections in terms of the process yield and productivity.     

一种疏水色谱填料的特性及应用的研究

 以交联壳聚糖为基质 ,正戊醛为配基 ,利用改进的方法制备了疏水作用色谱 (HIC)填料 ,并对该色谱填料的吸附行为和应用作了研究。结果表明 ,此类填料对蛋白质的吸附行为符合疏水相互作用理论 ,对α 淀粉酶的纯化活性回收率大于 80 %。     

疏水作用色谱介质的合成及色谱特性研究

利用国产大孔硅胶作基质合成了疏水填料。按照高效疏水作用色谱法,采用梯度洗脱方式分离了6种标准蛋白及唾液中α-淀粉酶和基因工程生产的γ-干扰素。柱子不可逆吸附小、被试验的α-淀粉酶和溶菌酶活性几乎定量被回收。应用合成的色谱填料研究了洗脱剂中盐浓度和温度对蛋白质保留行为的影响,论证了合成填料的色谱属性。     

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.     

Effects of thermal heterogeneity in hydrophobic interaction chromatography

Manipulating temperature and salt concentration can have a powerful effect on the separation effectiveness in hydrophobic interaction chromatography (HIC). However, use of temperature as an operating variable in large-scale applications may involve undesirable consequences such as radial heterogeneity of the column temperature. In this study non-ideal effects of heat transfer in HIC columns were analyzed. The radial temperature gradients were measured by thermocouples immersed in a bed packed into a preparative column. The column wall was either thermostatted by a water jacket or left under ambient conditions. The influence of ineffective column thermostatting and of heat losses on the radial temperature profiles was demonstrated and predicted by a model of heat dispersion in a packed bed. To analyze possible positive or negative effects of thermal heterogeneity on band propagation, non-isothermal chromatographic elution of a model protein (α-chymotrypsinogen A) was recorded under salt gradient conditions as well as at constant salt concentration. To predict temperature and concentration profiles a model of the column dynamics was used. The model accounted for kinetics of mass and heat transfer. A good agreement between experimental and simulated profiles was achieved. It was shown that by proper selection of the process conditions undesirable temperature effects can be avoided or controlled.     

Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography

Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, α-chymotrypsinogen A, β-lactoglobulin B, and holo α-lactalbumin, are studied on Sepharose™ HIC resins possessing assorted ligand chemistries and densities. α-Chymotrypsinogen, conformationally the most stable protein in the set, exhibits no change in solvent exposure at all the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed β-lactoglobulin B and holo α-lactalbumin range from −4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2–0.9 min⁻¹. While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.     

疏水层析蛋白质动力学与平衡过程的考察

疏水层析是分离生物大分子的常用技术之一，但对疏水层析中蛋白质吸附动力学和平衡过程的研究并不多见．本文对蛋白质疏水吸附动力学和平衡过程作了基本假设，并用实验进行了验证。制备了两种不同丁基密度的疏水琼脂糖介质，用其吸附牛血清白蛋白(BSA)以验证对疏水吸附动力学与平衡过程作的假设，考察了盐浓度及配基密度对蛋白质疏水吸附的影响．还对三种疏水性不同的蛋白质：核糖核酸酶、卵清蛋白和牛血清白蛋白的混合体系进行了分离性能的研究，获得了满意的分离效果．实验表明，蛋白质在疏水介质上的吸附动力学和平衡过程与所作假设相符，在实验条件下等温吸附线符合Langmuir吸附等温方程：研制的丁基琼脂糖疏水介质具有优良的使用性能。     

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.     

Protein adsorption on novel acrylamido-based polymeric ion-exchangers III. Salt concentration effects and elution behavior

The effect of salt concentration on the adsorption and desorption of BSA has been determined for a polymeric anion-exchanger based on acrylamido monomers. The material investigated possesses a high adsorption capacity at low salt concentration and the bound protein can be recovered quantitatively at high salt concentrations. The effects of salt on adsorption and desorption rates were evaluated from batch and shallow-bed experiments, and a model was developed to describe the data quantitatively. The adsorption capacity decreases as the salt concentration is increased, but both adsorption and desorption rates increase at higher salt concentrations. The predictability of the behavior of columns packed with this material was examined by comparing model predictions and experimental results obtained in laboratory columns. In general, a good agreement was obtained between predicted and experimental breakthrough and elution profiles, especially in shorter columns. Thus, the model allows a prediction of the effects of column length, mobile phase flow-rate, protein feed concentration, and salt concentration on dynamic capacity, productivity, and on the concentration of product fractions.     

Loading, stationary phase, and salt effects during hydrophobic interaction chromatography: alpha-lactalbumin is stabilized at high loadings

Amide hydrogen-deuterium exchange labeling has been used to study the effects of salt and protein loading on alpha-lactalbumin (BLA) stability during hydrophobic interaction chromatography (HIC). Stability in the adsorbed phase increased dramatically with increasing loading, and unfolding was nearly undetectable close to the resin saturation capacity. We also found that a butyl surface destabilized BLA more than a phenyl surface, despite the fact that BLA was bound more strongly on the phenyl surface. These observations have important implications for HIC process design and indicate that in some cases column capacity does not have to be sacrificed to preserve protein stability.     

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.     

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.     

Effect of pH changes on water release values in hydrophobic interaction chromatographic systems

The effect on pH on protein binding in HIC systems was investigated. Isocratic experiments were carried out to determine the capacity factors of various proteins as a function of temperature, pH and salt type. This paper presents a framework based on the Maxwell linkage function for estimating the number of released water molecules during the adsorption/desorption process due to a change of buffer pH. This approach also enables one to predict the effect of pH change on the water released values upon binding at any temperature condition. The results indicate that the total number of released water molecules (delta nu) for a pH change increased more on aromatic surfaces (phenyl Sepharose) than on aliphatic resins (butyl Sepharose). The results also indicate that the total number of released water molecules (deltanu) for a pH change increased with salt concentration and when changing from chaotropic to kosmotropic salts. The (deltanu) values also increased as the buffer pH approached the protein's pI, and decreased away from its pI. This work helps to establish a framework for the investigation of pH effects on protein selectivity in HIC systems.     

Altering efficiency of hydrophobic interaction chromatography by combined salt and temperature effects

The coupled effect of salt concentration and temperature on the retention behavior of proteins in hydrophobic interaction chromatography has been studied. The retention data of four model proteins, i.e., myoglobin, lysozyme, α-chymotrypsinogen and bovine serum albumin, have been acquired by isocratic experiments of chromatographic elution within the temperature range 5–25 °C at different ammonium sulphate concentrations in the mobile phase. The retention dependencies quantified as functions of the salt concentration and temperature have been exploited in designing the process of gradient elution. The propagation velocity of proteins under conditions of the step gradient of salt and temperature has been determined by use of the equilibrium theory. To evaluate kinetic effects accompanying the band propagation the transport-dispersive model has been employed. It has been shown that altering the propagation of the salt and temperature waves in a proper manner allows improving the separation efficiency. Moreover, manipulation of specific kinetics effects can also be exploited in protein separations.     

pH Transients in hydroxyapatite chromatography columns—Experimental evidence and phenomenological modeling

Hydroxyapatite (HAP) columns, widely used for chromatographic separation of proteins and other biomolecules because of their unique selectivity and ability to resolve complex mixtures, exhibit limited stability at acidic conditions requiring careful control of pH. Even with buffered solutions, however, unintended pH transients can occur when the salt concentration varies. For example, the pH temporarily decreases below the feed value when the salt concentration increases and increases above the feed value when the salt concentration is decreased. The intensity and duration of these transients depend on the particular buffer used and the magnitude of the salt concentration step, but in extreme cases the pH can drop by as much as 1.5 pH units creating conditions where the HAP stability is potentially compromised. This work examines the mechanisms leading to pH transients in HAP columns generated by salt steps. The pH excursions are similar to those observed for weak cation exchange columns, but are accompanied by a transient evolution of phosphate which temporarily decreases below the feed value when the salt concentration is increased and increases sharply when the salt concentration is reduced before returning to the feed value. A phenomenological model is developed to describe this behavior by considering the reversible uptake of sodium ions by the P-sites and binding of phosphate ions by the C-sites. The interplay of these two adsorption mechanisms results in complex pH patterns that are consistent with those observed experimentally. In addition to helping understand the underlying mechanisms, the model also provides a useful tool to predict the effects of different buffers and salt concentration and develop corrective measures that can reduce the intensity and duration of the pH transients such as the addition of unretained co-buffers.     

乙二醇-磷霉素改性氧化锆色谱固定相的制备及其在蛋白质分离中的应用

合成了分离蛋白质的乙二醇-磷霉素钠改性氧化锆高效液相色谱固定相, 通过漫反射红外光谱、元素分析等分析方法对该固定相进行了表征. 以溶菌酶、核糖核酸酶A、细胞色素C和糜蛋白酶四种标准碱性蛋白质为探针, 系统地考察了固定相的疏水相互作用色谱性能. 结果表明, 乙二醇-磷霉素改性氧化锆固定相对蛋白质有一定的保留, 表现出较高的分离选择性.     

Modeling of the salt effects on hydrophobic adsorption equilibrium of protein

A two-state protein model is proposed to describe the salt effects on protein adsorption equilibrium on hydrophobic media. This model assumes that protein molecules exist in two equilibrium states in a salt solution, that is, hydrated and dehydrated states, and only the dehydrated-state protein can bind to hydrophobic ligands. In terms of the two-state protein hypothesis and the steric mass-action theory, protein adsorption equilibrium on hydrophobic media is formulated by a five-parameter equation. The model is demonstrated with the adsorption of bovine serum albumin to Phenyl Sepharose gels as a model system. The effects of salt type (sodium chloride, sodium sulfate and ammonium sulfate) on the model parameters are discussed. Then, the model formulism is simplified in terms of the small magnitude of the protein dehydration equilibrium constant in the model. This simplification has returned the model derived on the basis of the two-state protein hypothesis to its original mechanism of salt effects on the hydrophobic adsorption of protein. This simplified model also creates satisfactory prediction of protein adsorption isotherms.