Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations. The simulations showed the separation depended on both the fluid residence time and the number of theoretical plates. However, when the number of theoretical plates was increased to ≥150, the simulations predicted that the separation performance of the column was not significantly improved. The approach described here could be used as a method to quantify acceptable height equivalent of a theoretical plate values for columns, and serve as a tool to understand how column packing quality impacts a given chromatographic separation prior to column scale-up, as well as during the monitoring of column lifetime in the manufacturing of large scale protein therapeutics.     

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.     

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.     

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.     

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.     

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.     

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.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

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.     

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.     

Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization

Chromatography is one of the key operations in the downstream processing of plasmid DNA (pDNA). However, the increased demand for highly purified pDNA experienced in recent years has made clear the need for alternative processes capable of retaining the advantages of conventional chromatography, such as selectivity, while providing increased throughput at a lower cost. The work presented in this article outlines the development and optimization of an alternative hydrophobic interaction membrane chromatography process for the purification of pDNA. The studies included the modification of functionalized membrane supports with a linear alkyl chain ligand and the testing of chromatographic performance of these membranes. Three modification procedures were tested and the membranes were screened for their capacity and selectivity. The modified membranes could separate the model plasmid pVAX1‐LacZ (6050 bp) from impurities in clarified Escherichia coli cell lysates (specifically RNA), with good resolution. Subsequent optimization of elution profiles with the best‐performing modified membrane, resulted in a high purification factor of 4.7, competitive with its bead process counterpart, and a plasmid yield of 73%.     

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.     

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.     

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.     

Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion-exchange resins

Immunoglobulin G is an important plasma protein with many applications in therapeutics and diagnostics, which can be purified effectively by ion exchange chromatography. The ligand densities and pore properties of ion-exchange resins have significant effects on the separation behaviors of protein, however, the understandings are quite limited. In this work, with bovine immunoglobulin as the model IgG, the adsorption isotherms and adsorption kinetics were investigated systematically with series of diethylaminoethyl ion-exchange resins with different ligand densities and pore sizes. The Langmuir equation and pore diffusion model were used to fit the experimental data. The influences of ligand density and pore size on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. The adsorption capacities increased with the increase of ligand density and the decrease of pore size, and an integrative parameter was proposed to describe the combined effects of ligand density and pore size. It was also found that the effective pore diffusion coefficient of the adsorption kinetics was influenced by pore sizes of resins, but was relatively independent on the ligand densities of resins. For a given protein, the ligand density and pore size should be optimized for improving the protein adsorption.     

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.     

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%.     

Direct recovery of recombinant nucleocapsid protein of Nipah virus from unclarified Escherichia coli homogenate using hydrophobic interaction expanded bed adsorption chromatography

A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2 mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPa s. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.     

氨基己酸为配基的新型弱阳离子交换/疏水双功能色谱固定相对蛋白质的分离纯化

以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水（WCX/HIC）双功能混合模式色谱固定相。该固定相配基具有一定的疏水性且含有羧基,在高盐浓度下表现为HIC的性质,可作为HIC固定相使用;在低盐浓度条件下表现为离子交换的性质,可作为WCX固定相使用。分别考察了该介质在WCX和HIC两种模式下对标准蛋白质的分离性能,并与商品柱进行比较。结果表明,所合成的WCX/HIC双功能固定相在WCX和HIC两种模式下对蛋白质均有较高的分离度和选择性,且分离能力与商品柱相当,两种模式下标准蛋白质的质量和活性回收率均大于93%,表明该柱具有“一柱二用”的功能,适于生物大分子的分离纯化。基于此双功能色谱柱构建的在线单柱二维液相色谱（2DLC-1C）可在60 min内实现8种蛋白质的快速分离。在70 min内完成了对蛋清中溶菌酶的二维纯化,纯度可达到98.3%。该技术中一根色谱柱可当作两根色谱柱使用,对蛋白质组学研究和重组蛋白药物的生产具有重要的应用价值。     

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.