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.     

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.     

Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems

A general rate model was employed in concert with a preferential interaction quadratic adsorption isotherm for the characterization of HIC resins and the prediction of solute behavior in these separation systems. The results indicate that both pore and surface diffusion play an important role in protein transport in HIC resins. The simulated and experimental solute profiles were compared for two model proteins, lysozyme and lectin, for both displacement and gradient modes of chromatography. Our results indicate that a modeling approach using the generate rate model and preferential interaction isotherm can accurately predict the shock layer response in both gradient and displacement chromatography in HIC systems. While pore and surface diffusion played a major role and were limiting steps for proteins, surface diffusion was seen to play less of a role for the displacer. The results demonstrate that this modeling approach can be employed to describe the behavior of these non-linear HIC systems, which may have implications for the development of more efficient preparative HIC separations.     

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.     

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.     

3D structure-based protein retention prediction for ion-exchange chromatography

The interest in understanding fundamental mechanisms underlying chromatography drastically increased over the past decades resulting in a whole variety of mostly semi-empirical models describing protein retention. Experimental data about the molecular adsorption mechanisms of lysozyme on different chromatographic ion-exchange materials were used to develop a mechanistical model for the adsorption of lysozyme onto a SP Sepharose FF surface based on molecular dynamic simulations (temperature controlled NVT simulations) with the Amber software package using a force-field based approach with a continuum solvent model. The ligand spacing of the adsorbent surface was varied between 10 and 20 Å. With a 10 Å spacing it was possible to predict the elution order of lysozyme at different pH and to confirm in silico the pH-dependent orientation of lysozyme towards the surface that was reported earlier. The energies of adsorption at different pH values were correlated with isocratic and linear gradient elution experiments and this correlation was used to predict the retention volume of ribonuclease A in the same experimental setup only based on its 3D structure properties. The study presents a strong indication for the validity of the assumption, that the ligand density of the surface is one of the key parameters with regard to the selectivity of the adsorbent, suggesting that a high ligand density leads to a specific interaction with certain binding sites on the protein surface, while at low ligand densities the net charge of the protein is more important than the actual charge distribution.     

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.     

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。     

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.     

Retention model of protein for mixedmode interaction mechanism in ion exchange and hydrophobic interaction chromatography

A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a "U" shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.     

Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution.     

Parallel pore and surface diffusion of levulinic acid in basic polymeric adsorbents

The equilibrium and kinetics of levulinic acid (LA) adsorption on two basic polymeric adsorbents, 335 (highly porous gel) and D315 (macroreticular), were investigated. Experimental adsorption rates in batch stirred vessels under a variety of operating conditions were described successfully by the parallel pore and surface diffusion model taking into account external mass transfer and nonlinear Toth isotherm. The film-pore diffusion model was matched with the rate data and the resulting apparent pore diffusivities were strongly concentration-dependent and approached to a constant value for 335 adsorbent. Thus, the constant value was taken as the accurate pore diffusivity, while the pore diffusivity in D315 was estimated from the particle porosity. The surface diffusivities decreased with increasing initial bulk concentration for both adsorbents. The inverse concentration dependence was correlated reasonably well to the change of isosteric heat of adsorption as amount adsorbed.     

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.     

Determinants of protein retention characteristics on cation-exchange adsorbents.

There are currently a large number of commercially available strong and weak cation-exchange adsorbents for preparative protein purification, typically prepared by coupling charged ligands to a mechanically rigid porous bead. Because of the diverse chemical nature of the base matrix (carbohydrate, synthetic polymer, inorganic) and the coupling and ligand chemistry, cation-exchange adsorbents from different suppliers can differ substantially in chemical surface properties and physical structure. The differences in chemical properties can be in ionic capacity, hydrophobicity, the presence of hydrogen bond donors/acceptors, and the nature of the charged functional groups. In order to probe the effects of these factors on protein affinity, the isocratic retention of a set of model proteins was examined on a set of cation-exchange adsorbents to obtain a quantitative assessment of retention differences between adsorbents. Two adsorbent factors were found to be the dominant determinants of overall protein retention: the anion type and the adsorbent pore size distribution. Protein retention on strong cation-exchangers was found to be greater than that on corresponding weak cation-exchangers. Protein retention was increased on adsorbents with pore size distributions that include significant amounts of pore space with dimensions similar to those of the protein solute.     

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.     

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.     

Role of Physical Forces in Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is a new non-biospecific liquid chromatography method for the separation of proteins, and other biological macromolecules; the mobile phases are aqueous and the adsorbents are agarose beads coated with ionogenic, or non-ionogenic, hydrocarbonaceous ligands. A non-traditional interpretation is given here for the mechanisms of retention and elution in HIC in terms of the several physical forces between the protein and the adsorbent, chiefly the van der Waals attraction (comprising dispersion, orientation and induction) and the electrostatic double layer interaction. From a qualitative analysis of the hydrogen-bond and the structural features of water, it is shown here that the role of the alkyl ligands on the adsorbent, the lyotropic salt effects and the effect of additives to the mobile phase such as ethylene glycol can all be unifyingly represented in the Hamaker coefficient of the van der Waals attraction between protein and adsorbent in water. An increase in the number and length of alkyl ligands, and the addition of structure-making (“salting-out”) salts at high ionic strengths increase the latter attraction while the addition of structure-breaking “salting-in” salts or organic solvents such as ethylene glycol decreases the attraction. The potentials corresponding to the different physical forces add up to the total interaction potential. The shapes of the total interaction potential relevant to HIC are identified. Coefficients of adsorption and desorption are shown to be related to this potential and the high sensitivity of the latter to its parameters such as the Hamaker coefficient, is illustrated. Retention, elution and the natures of the different fractions into which a protein mixture may be separated by HIC are visualized in terms of the interaction potential.

Numerous experimental reports in HIC are classified, tabulated, and in certain cases, discussed in detail. Experimental evidence is presented for the application of ionic strength manipulations, in the low ionic strength range (electrostatic effects), in the high ionic strength range (lyotropic salt effects) as well as for the combined use of low and high ionic strength effects, retention onto adsorbents with no ligands as well as onto adsorbents with alkyl ligands of various chain lengths and number densities, temperature effects, and the effects of heterogeneities of proteins and adsorbents. Applications and variants of HIC are cited. In total, the paper summarizes various types of HIC experimental facts and explains most of them in a simple, unifying fashion.     

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.