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.     

Specific salt effects in hydrophobic interaction chromatography of proteins

Summary It has been noted in the literature that certain salts enter into specific interaction with proteins. As a result of this, they may act as salting-in agents. We have investigated the effect of magnesium chloride which is known to possess such unusual properties on the retention of proteins in hydrophobic-interaction chromatography. First the retention behaviour of amino acids and small peptides having a wide polarity range was studied on reversed-phase columns using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂, in wide the concentration ranges. For less polar eluites plots of the logarithmic retention factors against the salt concentration were found to be linear, whereas the more polar species showed irregular behavior. The retention of a wide range of proteins was measured on a TSK Phenyl-5-PW column using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂ at different concentrations.The salt-mediated retention was regular with (NH₄)₂ SO₄ and MgSO₄ although MgSO₄ showed a lesser effect than that predicted by the surface-tension increment. The effect of MgCl₂ was quite irregular: the retention factors either increased or decreased or remained unchanged depending on the protein. These results corroborate earlier observations regarding the particular effect of MgCl₂ and suggest the modulation of selectivity in hyrophobic-interaction chromatography by the addition of MgCl₂ to the eluent.     

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.     

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.     

Polymer-based monolithic microcolumns for hydrophobic interaction chromatography of proteins

Monolithic capillary columns for hydrophobic interaction chromatography (HIC) have been prepared by thermally initiated, single-step in situ polymerization of mixtures of monovinyl monomers including butyl methacrylate and/or 2-hydroxyethyl methacrylate, with a divinyl crosslinker glycerol dimethacrylate or 1,4-butanediol dimethacrylate using two different porogen systems. Two porogenic solvent mixtures were used; one "hydrophilic", consisting of water, butanediol, and propanol, and one "hydrophobic," comprising dodecanol and cyclohexanol. The porous structures of the monoliths were characterized and their performance was demonstrated with a separation of a mixture of myoglobin, ribonuclease A, and lysozyme under conditions typical of HIC.     

Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography

This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 μm pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%.     

Resolution enhancement in hydrophobic interaction chromatography via electrostatic interactions

In this work, a new type of hydrophobic stationary phase that provide electrostatic interactions with analytes was developed by bonding β-phenylethylamine as a functional ligand to silica. This stationary phase can separate proteins with similar hydrophobicity that traditional hydrophobic resins cannot. Hen egg white was separated to examine the selectivity. The results show that the introduced electrostatic interactions are an important factor for the resolution enhancement and the new resin could have important applications in separation and purification of biological macromolecules.     

Nylon-6 capillary-channeled polymer (C-CP) fibers as a hydrophobic interaction chromatography stationary phase for the separation of proteins

Nylon-6 capillary-channeled polymer (C-CP) fibers are used as the stationary phase for the hydrophobic interaction chromatography (HIC) separation of a synthetic protein mixture composed of ribonuclease A, lysozyme, and holotransferrin. Nylon is a useful polymer phase for HIC as it has an alkyl backbone, while the amide functionality is hydrophilic (in fact ionic) in nature. The combination of a nonporous polymer surface of the fiber phases and high column permeability yields very efficient mass transfer characteristics, as exhibited by narrowing of peak widths with increases in mobile phase linear velocity. Retention factors and resolution were evaluated at flow rates ranging from 0.5 to 9 mL/min (linear velocities of ca. 2 to 15 mm/s) and at gradient slopes between 3.3 and 20 %B/min. Optimum resolution was achieved by employing fast flow rates (9 mL/min) and slow gradients (3 %B/min), also resulting in the highest peak capacities.     

Effect of phenyl sepharose ligand density on protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

In the large-scale manufacturing and purification of protein therapeutics, multiple chromatography adsorbent lots are often required due to limited absorbent batch sizes or during replacement at the end of the useful column lifetime. Variability in the adsorbent performance from lot to lot should be minimal in order to ensure that consistent product purity and product quality attributes are achieved when a different lot or lot mixture is implemented in the process. Vendors of chromatographic adsorbents will often provide release specifications, which may possess a narrow range of acceptable values. Despite relatively narrow release specifications, the performance of the adsorbent in a given purification process could still vary from lot to lot. In this case, an alternative use test (one that properly captures the lot to lot variability) may be required to determine an acceptable range of variability for a specific process. In this work, we describe the separation of therapeutic protein monomer and aggregate species using hydrophobic interaction chromatography, which is potentially sensitive to adsorbent lot variability. An alternative use test is formulated, which can be used to rapidly screen different adsorbent lots prior to implementation in a large-scale manufacturing process. In addition, the underlying mechanism responsible for the adsorbent lot variability, which was based upon differences in protein adsorption characteristics, was also investigated using both experimental and modeling approaches.     

Polyethylene glyco-bonded phases for protein separation by high-performance hydrophobic interaction chromatography

Summary This paper further investigates the effects of silica base pore size and the molecular weight of polyethylene glycol (PEG) ligands on the coverage of PEG-bonded phases, as well as the resolution of protein separation in high-performance hydrophobic interaction chromatography (HPHIC). The results demonstrate that among the PEG-bonded phases examined in this study, the bonded phase coupled PEG-1500 on LiChrospher 500 silica exhibited the best resolution in protein separation.     

Recombinant protein purification using gradient assisted simulated moving bed hydrophobic interaction chromatography. Part II: process design and experimental validation

In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated.     

Methods of calculating protein hydrophobicity and their application in developing correlations to predict hydrophobic interaction chromatography retention

Hydrophobic interaction chromatography (HIC) is a key technique for protein separation and purification. Different methodologies to estimate the hydrophobicity of a protein are reviewed, which have been related to the chromatographic behavior of proteins in HIC. These methodologies consider either knowledge of the three-dimensional structure or the amino acid composition of proteins. Despite some restrictions; they have proven to be useful in predicting protein retention time in HIC.     

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.     

Purification and analysis of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography

We discuss the purification of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography. The hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono–, di–, and tri–), mono‐PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS‐PAGE, dynamic light scattering, and SEC combined with multi‐angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono‐PEGylated HSA. A scaled‐down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins.     

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.     

Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.     

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.     

Hydrophobic interaction chromatography at low salt concentration for the capture of monoclonal antibodies

We evaluated hydrophobic interaction chromatography (HIC) at low salt concentration for the capture of proteins from feed stocks by using monoclonal antibodies as model samples. It was indicated that the HIC at low salt concentration on critical hydrophobicity supports has a potential for capturing hydrophobic monoclonal antibodies directly from large volumes of feed stocks and recovering bound monoclonal antibodies in high yield. On the other hand, the HIC at low salt concentration did not seem so useful for the capture of weakly hydrophobic monoclonal antibodies. The recovery of weakly hydrophobic monoclonal antibodies from columns packed with critical hydrophobicity supports was not quantitative and significantly decreased as the residence time of the monoclonal antibodies in the columns became longer.     

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.