Hydrophobic interaction chromatography of proteins IV. Kinetics of protein spreading

Adsorption of proteins on surfaces of hydrophobic interaction chromatography media is at least a two-stage process. Application of pure protein pulses (bovine serum albumin and beta-lactoglobulin) to hydrophobic interaction chromatography media yielded two chromatographic peaks at low salt concentrations. At these salt concentrations, the adsorption process is affected by a second reaction, which can be interpreted as protein spreading or partial unfolding of the protein. The kinetic constants of the spreading reaction were derived from pulse response experiments at different residence times and varying concentrations by applying a modified adsorption model considering conformational changes. The obtained parameters were used to calculate uptake and breakthrough curves for spreading proteins. Although these parameters were determined at low saturation of the column, predictions of overloaded situations could match the experimental runs satisfactorily. Our findings suggest that proteins which are sensitive to conformational changes should be loaded at high salt concentrations in order to accelerate the adsorption reaction and to obtain steeper breakthrough curves.     

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.     

Alpha-lactalbumin tertiary structure changes on hydrophobic interaction chromatography surfaces

Hydrogen exchange (HX) detected by mass spectrometry (MS) was used to analyze the structure of calcium-free alpha-lactalbumin, a model protein with marginal stability. Two chromatographic peaks were observed from samples of pure protein eluted from SOURCE phenyl hydrophobic interaction chromatography (HIC) media. Whole-protein HX measurements showed that the less-retained peak had solvent exposure similar to native protein in the absence of the HIC surface while the retained protein was nearly, although not fully, solvent exposed. The formation of these two peaks was kinetically limited. The protein also refolded successfully following elution. In addition, proteolytic fragmentation was used to analyze HX at the peptide level. This approach revealed that helix C was the most stable region of alpha-lactalbumin under native conditions and in the flow-through peak. Helix C also formed the core of residual native structure in the partially unfolded protein in the retained peak. The results suggest that residues that are most solvent accessible under native conditions may be those most likely to unfold upon adsorption.     

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.     

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.     

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.     

Mixed retention mechanism of proteins in weak anion-exchange chromatography

Using four commercial weak anion-exchange chromatography (WAX) columns and 11 kinds of different proteins, we experimentally examined the involvement of hydrophobic interaction chromatography (HIC) mechanism in protein retention on the WAX columns. The HIC mechanism was found to operate in all four WAX columns, and each of these columns had a better resolution in the HIC mode than in the corresponding WAX mode. Detailed analysis of the molecular interactions in a chromatographic system indicated that it is impossible to completely eliminate hydrophobic interactions from a WAX column. Based on these results, it may be possible to employ a single WAX column for protein separation by exploiting mixed modes (WAX and HIC) of retention. The stoichiometric displacement theory and two linear plots were used to show that mechanism of the mixed modes of retention in the system was a combination of two kinds of interactions, i.e., nonselective interactions in the HIC mode and selective interactions in the IEC mode. The obtained U-shaped elution curve of proteins could be distinguished into four different ranges of salt concentration, which also represent four retention regions.     

Hydrophobic interaction chromatography of proteins. I. Comparison of selectivity

Currently, the selection of a hydrophobic interaction chromatography (HIC) sorbent for protein separation purposes is entirely based on empirical means. An attempt was made to characterize different HIC sorbents from various manufacturers. The selectivity was determined by isocratic pulse experiments of a set of reference proteins and an algorithm was developed to classify the sorbents according to their selectivity and hydrophobicity. The obtained semi-quantitative parameters take into account the dependence of salt on adsorption. The sorbent characteristics evaluated with the model proteins were compared to the separation of a real feedstock. A good agreement was achieved between the developed evaluation procedure and the separation behaviour of the real feed stock.     

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。     

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.     

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.     

Hydrophobic interaction chromatography of proteins. I. The effects of protein and adsorbent properties on retention and recovery

The contributions of protein and adsorbent properties to retention and recovery were examined for hydrophobic interaction chromatography (HIC) using eight commercially available phenyl media and five model proteins (ribonuclease A, lysozyme, alpha-lactalbumin, ovalbumin and BSA). The physical properties of the adsorbents were determined by inverse size exclusion chromatography (ISEC). The adsorbents examined differ from each other in terms of base matrix, ligand density, porosity, mean pore radius, pore size distribution (PSD) and phase ratio, allowing systematic studies to understand how these properties affect protein retention and recovery in HIC media. The proteins differ in such properties as adiabatic compressibility and molecular mass. The retention factors of the proteins in the media were determined by isocratic elution. The results show a very clear trend in that proteins with high adiabatic compressibility (higher flexibility) were more strongly retained. For proteins with similar adiabatic compressibilities, those with higher molecular mass showed stronger retention in Sepharose media, but this trend was not observed in adsorbents with polymethacrylate and polystyrene divinylbenzene base matrices. This observation could be related to protein recovery, which was sensitive to protein flexibility, molecular size, and conformation as well as the ligand densities and base matrices of the adsorbents. Low protein recovery during isocratic elution could affect the interpretation of protein selectivity results in HIC media. The retention data were fitted to a previously published retention model based on the preferential interaction theory, in terms of which retention is driven by release of water molecules and ions upon protein-adsorbent interaction. The calculated number of water molecules released was found to be statistically independent of protein retention strength and adsorbent and protein properties.     

疏水作用色谱填料研究进展

疏水作用色谱法（HIC）是一种有效的蛋白质分离纯化方法。综述了HIC填料的基质和配基的发展过程,举例说明了不同HIC填料对蛋白质的分离纯化效率,指出了制备型HIC填料的现状及其发展趋势。     

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.     

Protein stability and structure in HIC: hydrogen exchange experiments and COREX calculations

Hydrogen exchange mass spectrometry (HXMS) coupled to proteolytic digestion has been used to probe the conformation of bovine β-lactoglobulin (BLG), bovine α-lactalbumin (BLA), and human serum albumin (HSA) in solution and while adsorbed to the hydrophobic interaction chromatography media Phenyl Sepharose 6FF. All three proteins show evidence of EX1 exchange kinetics, indicating a loss of stability on the surface. HX protection patterns for all three proteins also indicate that the unfolded form is only partially solvent exposed. The hydrogen-deuterium exchange patterns of BLG and BLA on the surface suggest a structure that resembles each protein's respective solution phase molten globule state. The low stability of Domain II of HSA observed on Phenyl Sepharose 6FF also suggests a link to solution stability because Domain II is frequently cited as the least stable domain in solution unfolding pathways. COREX, an algorithm used to compute protein folding stabilities, correctly predicts solution hydrogen-deuterium exchange patterns for BLG and offers insight into its adsorbed phase stabilities but is unreliable for BLA predictions. The results of this work demonstrate a link between solution-phase local stability patterns and the nature of partially unfolded states that proteins can adopt on HIC surfaces.     

Prediction of retention time of cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan-proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Phitagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE<0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE<0.022) for the two tagged cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose-2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of cutinases tagged with hydrophobic peptides in HIC.     

疏水作用色谱中流动相组成对溶质保留行为的影响

 首次研究了疏水作用色谱 (HIC)中芳香醇同系物在不同种类盐流动相中的保留行为。以计量置换保留模型中的参数Z分析了HIC中小分子与生物大分子保留行为的差别 ,以及不同流动相组成对两种类型溶质的洗脱范围及洗脱能力的影响。与反相色谱相似 ,芳香醇在HIC中的保留仍存在同系物规律。比较了小分子和生物大分子在不同盐溶液中的Z值变化 ,表明流动相中的盐仅改变小分子与固定相的水合程度 ,而对生物大分子 ,除改变其和固定相水合程度外 ,还会影响生物大分子与固定相接触区的分子构象     

Spreading of proteins and its effect on adsorption and desorption kinetics

The kinetics of adsorption of lysozyme and alpha-lactalbumin from aqueous solution on silica and hydrophobized silica has been studied. The initial rate of adsorption of lysozyme at the hydrophilic surface is comparable with the limiting flux. For lysozyme at the hydrophobic surface and alpha-lactalbumin on both surfaces, the rate of adsorption is lower than the limiting flux, but the adsorption proceeds cooperatively, as manifested by an increase in the adsorption rate after the first protein molecules are adsorbed. At the hydrophilic surface, adsorption saturation (reflected in a steady-state value of the adsorbed amount) of both proteins strongly depends on the rate of adsorption, but for the hydrophobic surface no such dependency is observed. It points to structural relaxation ("spreading") of the adsorbed protein molecules, which occurs at the hydrophobic surface faster than at the hydrophilic one. For lysozyme, desorption has been studied as well. It is found that the desorbable fraction decreases after longer residence time of the protein at the interface.