Glycerol-Assisted Hydrophobic Interaction Chromatography Improving Refolding of Recombinant Human Granulocyte Colony-Stimulating Factor

Efficient refolding of recombinant proteins in the forms of inclusion bodies at higher concentration remains challenging. Here, we report a strategy of a dual-gradient hydrophobic interaction chromatography (HIC) mode to refold recombinant human granulocyte colony-stimulating factor from its inclusion bodies at high protein concentration. The strategy was taken to meet the demand of dynamic refolding proceeding by gradually decrease the denaturant (guanidine-HCl) concentration and gradually increase the hydrophilicity of media (column of Poros PE 20) with glycerol as additive to provide a mild refolding surroundings. Compared with dilution method, this dual-gradient HIC process gave about 8.5-fold of increase in specific activity and 30% increase in soluble protein recovery. Furthermore, much higher protein concentration could be obtained at the same time.     

Two-step recovery process for tryptophan tagged cutinase: interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)4 was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to >95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process.     

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.     

Continuous chromatographic protein refolding

Column-based protein refolding requires a continuous processing capability if reasonable quantities of protein are to be produced. A popular column-based method, size-exclusion chromatography (SEC) refolding, employs size-exclusion matrices to separate unfolded protein from denaturant, thus refolding the protein. In this work, we conduct a comparison of SEC refolding with refolding by batch dilution, using lysozyme as a model protein. Lysozyme refolding yield was found to be extremely sensitive to the chemical composition of the refolding buffer and particularly the concentration of dithiothreitol (DTT) introduced from the denatured protein mixture. SEC refolding was not adversely affected by DTT carry-over as small contaminants in the denatured solution are separated from protein during the refolding operation. We also find that, contrary to previous reports, size-exclusion refolding on batch columns leads to refolding yields slightly better than batch dilution refolding yields at low protein concentrations but this advantage disappears at higher protein concentrations. As batch-mode chromatography would be the limiting step in a column based refolding downstream process, the batch column refolding method was translated to a continuously operating chromatography system (preparative continuous annular chromatography, P-CAC). It was shown that the P-CAC elution profile is similar to that of a stationary column, making scale-up and translation to P-CAC relatively simple. Moreover, it was shown that high refolding yields (72%) at high protein concentration (>1 mg ml(-1)) could be obtained.     

Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.     

Chromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191-345 column

Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-gamma) was carried out on a chromatographic column packed with immobilized minichaperone. The effects of salt concentration, urea concentration gradient, elution flow rate and protein loading on the refolding efficiency were investigated. The results indicated that immobilized sht GroEL191-345 chromatography was an effective protocol for the refolding of rhIFN-gamma. When loading 100 microl denatured rhIFN-gamma (17.8 mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74 x 10(6)IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity.     

Refolding and purification of rhNTA protein by chromatography

RhNTA protein is a new thrombolytic agent which has potential medicinal and commercial value. Protein refolding is a bottleneck for large‐scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. The denatured rhNTA protein was refolded by an improved size‐exclusion chromatography refolding process achieved by combining an increasing arginine gradient and a decreasing urea gradient (two gradients) with a size‐exclusion chromatography refolding system. The refolding of denatured rhNTA protein showed that this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial rhNTA protein concentration up to 20 mg/mL. After refolding by two‐gradient size‐exclusion chromatography refolding processes, the refolded rhNTA was purified by ion‐exchange and affinity chromatography. The purified rhNTA protein showed one band in SDS‐PAGE and the specific activity of purified rhNTA protein was 110,000 U/mg. Copyright © 2008 John Wiley & Sons, Ltd.     

Lysozyme refolding with immobilized GroEL column chromatography

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5 M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, there existed an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3 ml of the lysozyme (1 mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54 mg per 1 gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12 mg per 1 gel per h), indicating the advantage of the present system for producing a high throughput in protein refolding operations.     

Effects of thermal heterogeneity in hydrophobic interaction chromatography

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

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption

Protein refolding at high concentrations always leads to aggregation, which limits commercial application. An ion-exchange chromatography process with gradient changes in urea concentration and pH was developed to refold denatured lysozyme at high concentration. After adsorption of the denatured protein onto an ion-exchange medium, elution was carried out in combination with a gentle decrease in urea concentration and elevation of pH. Protein would gradually refold along the column with high activity yield. Denatured and reduced lysozyme at 40 mg/ml was loaded into a column filled with SP Sepharose Fast Flow, resulting in 95% activity recovery and 98% mass yield within a short period of time.     

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

A novel dual‐retention mechanism mixed‐mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high‐salt‐concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low‐salt‐concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion‐exchange chromatography and HIC columns. The results indicated that the novel dual‐retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a ‘2D column’. In addition, the mixed retention mechanism of proteins on this ‘2D column’ was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual‐retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this ‘2D column’, a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of degradation mechanisms by portable Raman spectroscopy and thermodynamic speciation: the wall painting of Santa María de Lemoniz (Basque Country, North of Spain)

To characterise the polymeric properties of processed lignins, a new method has been developed using hydrophobic interaction chromatography (HIC). This method separates the lignin polymers into fractions based on differences in hydrophobicity using low pressure liquid chromatography (LPLC). The hydrophobic column material consists of monodisperse polystyrene/divinylbenzene beads. An elution gradient was prepared monitoring the electrolyte concentration and pH. Citric acid buffer, containing ammonium sulphate that promotes adsorption to the column material, was used as mobile phase in a step-wise gradient together with ethanol (20/80% (v/v) ethanol/water, pH 12) and isopropanol (40/60% (v/v) isopropanol/water, pH 12). Depending on eluent composition, the degree of elution was 94% or higher. With the HIC method developed, lignosulphonates and kraft lignins were separated into seven distinctive peaks according to hydrophobicity.     

Separation and determination of polyethylene glycol fatty acid esters in cosmetics by a reversed-phase HPLC/ELSD

A comprehensive analytical method was established for the separation of polyethylene glycol (PEG) stearates according to the distribution of ethylene oxide (EO) and subsequent determination of the surfactants in cosmetic samples by using a high-performance liquid chromatography–evaporative light scattering detection. Separation of the PEG stearates comprising approximately up to 82 EO adducts was performed on a reversed-phase YMC-Pack C₈ column using water–acetonitrile gradient elution. The PEG oligomers were separated in order of the increasing number of EO adducts. Quantitation of the PEG fatty acid esters, which was separated as single peak per each component, was performed by chromatography on a reversed-phase Wakosil 10 C₁₈ column using water–methanol gradient elution. The standard curve to quantify the PEG stearates was constructed by the log–log plot, which showed good linearity with the correlation coefficients (R²) 0.998 and more. Working range, repeatability, limit of detection and recovery were acceptable for analysis of the surfactants in cosmetic products. The analytical methods were applied to characterize the PEG stearates according to the EO distributions, then to quantify the surfactants in cosmetic products.     

Purification of antibody heteropolymers using hydrophobic interaction chromatography

A heteropolymer (HP) is a unique dual antibody conjugate composed of specific, chemically cross-linked monoclonal antibodies (mAbs). In this study we have demonstrated that HPs can be purified using hydrophobic interaction chromatography (HIC). Two propyl HIC resins; [PolyPropyl A and EMD Fractogel Propyl (S)] were evaluated in this study. Phosphate buffers, pH 6.5 containing ammonium sulfate or sodium sulfate were used to bind the HP to the column. A descending sulfate gradient or step gradient was used to elute the bound HP species from the column. The HP reaction mixture typically contains multiple conjugated HP species, as well as unreacted monomer mAbs. Conjugated HP product was successfully separated from unreacted antibody monomers with both propyl resins using buffers with ammonium sulfate. There was no monomer separation from HP using buffers with sodium sulfate. The purification processes, presented in this study allows the non-cross-linked antibodies to pass through the column without being bound to the resin, while the cross-linked antibodies (the HP product) bound to the column were subsequently eluted by decreasing the ammonium sulfate concentration in the running buffer. HP product was efficiently separated from free mAbs using Propyl HIC resins at both analytical and preparative scales.     

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.     

One‐step refolding and purification of recombinant human tumor necrosis factor‐α (rhTNF‐α) using ion‐exchange chromatography

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic‐based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor‐α (rhTNF‐α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF‐α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF‐α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10⁸ IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF‐α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF‐α refolding in terms of specific bioactivity and mass recovery. Copyright © 2014 John Wiley & Sons, Ltd.     

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Protein‐folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high‐performance size‐exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low‐urea gradient‐elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS–18% PAGE, Matrix‐Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI‐TOF‐MS) and the biological activity assay by HP‐RPLC. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparation of phenylboronic acid functionalized cation-exchange monolithic columns for protein separation and refolding

In this study, we described a simple and effective modification procedure to prepare poly (methacrylate-co-ethylene glycol dimethacrylate) monolithic columns functionalized with 3-aminophenylboronic acid. The column morphology, pore size and specific surface area of the fabricated monolith were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, and mercury intrusion porosimeter, respectively. The frontal analysis was carried out for dynamic loading capacity of the model protein on the modified column. The chromatographic performance of the cation-exchange monolith was evaluated through separating a mixture of five proteins such as lysozyme, cytochrome c, ribonuclease A, trypsin and bovine serum albumin and one-step purification of lysozyme from egg whites, and the expected results were obtained. In addition, the functionalized column was used to refold ribonuclease A and cytochrome c, and this procedure was monitored by circular dichroism and fluorescence spectroscopy. Compared with the conventional dilution refolding method, the ion-exchange chromatography refolding method developed here is more effective for specific bioactivity recovery.     

Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55% after 40 h incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling.