Assessment of COMT isolation by HIC using a dual salt system and low temperature

Sodium citrate (SC) and low temperatures between 7 and 5°C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mm AS/200 mm SC and 25 mm AS/25 mm SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions. Copyright © 2009 John Wiley & Sons, Ltd.     

Hydrophobic interaction fast protein liquid chromatography of milk proteins

Bovine whey proteins and caseins were separated by hydrophobic interaction chromatography with the new Pharmacia fast protein liquid chromatography column, phenyl-Superose. Total casein was separated using a decreasing gradient of 0.8 to 0.05 M sodium phosphate and a constant 3.75 M urea concentration at pH 6.0. The order of elution of caseins was beta less than gamma, alpha s2 less than kappa less than alpha s1, and beta-casein was always eluted first. Whey proteins were separated with a decreasing salt gradient of 1.5 to 0 M ammonium sulphate in 0.05 M sodium phosphate at pH 7.0. The order of elution was beta-lactoglobulin less than bovine serum albumin less than immunoglobulin less than alpha-lactalbumin. The elution order of proteins from the column did not correlate with the calculated average hydrophobicities but the method was considered to be a measure of the "effective" hydrophobicity of proteins and therefore of more use for attempting to relate hydrophobicity to functional properties of proteins. The method shows significant advantages over conventional techniques allowing rapid optimization of elution conditions and reducing run times from 24 h or more to less than 2 h.     

Hydrophobicity gradient columns for the separation of trypsin inhibitor by hydrophobic interaction chromatography at low salt concentration

We investigated hydrophobicity gradient columns composed of two columns packed with supports of different hydrophobicities in order to save time in protein separation by hydrophobic interaction chromatography at low salt concentration using a crude sample of trypsin inhibitor as a model sample. One of the two hydrophobicity gradient columns was packed with a support whose hydrophobicity was critically controlled for target protein (trypsin inhibitor) and the other was packed with a support which was less hydrophobic than the critically controlled hydrophobicity support. It was found that the hydrophobicity gradient columns are useful to separate samples containing impurities of a wide range of hydrophobicities within a reasonable time.     

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.     

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.     

Comparison and evaluation of HIC columns of different hydrophobicity

Summary Retention and selectivity in hydrophobic interaction chromatography (HIC) depend both on the type of stationary phase and on the mobile phase. In the last few years various high performance packing materials and columns have been introduced for HIC resulting in a range of different retentions and selectivity. We have investigated the effect of the stationary phase on the retention of various proteins. The retention of some solutes of different hydrophobicities were measured on three commercial HIC columns (TSK-Phenyl, Synchropack-Propyl, CAA-HIC) under isocratic conditions using water-methanol mixtures as eluent. The log k_w values determined according to the literature were devalues determined according to the literature were dependent on the type and structure of the stationary phase and indicated a much less hydrophobic character for these columns than that obtained for reversed phase columns. Gradient separations were then carried out on a standard protein mixture using ammonium sulfate and sodium citrate to change the gradient time. In order to compare the effect of the stationary phase and the two salts investigated apparent capacity factors (k_g) were determined and plotted against the gradient time obtained for the three columns in the two eluent system. It was shown that the type of stationary phase had a significant effect on the retention of proteins. In addition, the effect of the mobile phase composition, i.e. salt type, was considerably different on the various stationary phases. In order to exploit the potential of HIC to modulate selectivity for the separation of proteins, the combined effect of the stationary phase and the type of salt should be taken into account.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Arginine improves protein elution in hydrophobic interaction chromatography. The cases of human interleukin-6 and activin-A

The effects of arginine on protein binding and elution in hydrophobic interaction chromatography (HIC) were examined using recombinant human interleukin-6 (IL-6) and activin-A. Binding of IL-6 in the presence of ammonium sulfate (AS) was tested using low- and high-substituted phenyl-sepharose. While inclusion of arginine during loading of IL-6 resulted in incomplete binding to the low-substituted phenyl-sepharose, binding was complete to the high-substituted phenyl-sepharose. Arginine facilitated elution of IL-6 from both columns. These results demonstrate that arginine weakens hydrophobic interactions between IL-6 and the phenyl-sepharose. More drastic results were obtained using activin-A, which showed undetectable recovery from phenyl-sepharose. Although no apparent elution of activin-A was observed from butyl-sepharose in aqueous buffer alone, the addition of arginine to the buffer resulted in partial elution recovery and, together with ethanol, resulted in greatly improved recovery of the protein. Two arginine derivatives, acetylarginine and agmatine, were also effective. These results show that arginine improves protein elution in HIC.     

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.     

Altering efficiency of hydrophobic interaction chromatography by combined salt and temperature effects

The coupled effect of salt concentration and temperature on the retention behavior of proteins in hydrophobic interaction chromatography has been studied. The retention data of four model proteins, i.e., myoglobin, lysozyme, α-chymotrypsinogen and bovine serum albumin, have been acquired by isocratic experiments of chromatographic elution within the temperature range 5–25 °C at different ammonium sulphate concentrations in the mobile phase. The retention dependencies quantified as functions of the salt concentration and temperature have been exploited in designing the process of gradient elution. The propagation velocity of proteins under conditions of the step gradient of salt and temperature has been determined by use of the equilibrium theory. To evaluate kinetic effects accompanying the band propagation the transport-dispersive model has been employed. It has been shown that altering the propagation of the salt and temperature waves in a proper manner allows improving the separation efficiency. Moreover, manipulation of specific kinetics effects can also be exploited in protein separations.     

Surfactant-mediated hydrophobic interaction chromatography of proteins: gradient elution

Addition of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulphonate (CHAPS) to mobile phases in gradient elution hydrophobic interaction chromatography (HIC) on SynChropak Propyl causes changes in observed elution times for nine globular proteins. The nine proteins showed different percentage reductions in capacity factor, k', demonstrating the ability of CHAPS to change the selectivity of the separations. Three basic types of gradient experiments have been explored for surfactant-mediated gradient elution HIC. Type I gradients are conducted with constant salt and variable surfactant concentration. Type II gradients with variable salt and constant surfactant concentration, and Type III gradients with variable salt and surfactant concentrations. By the criterion of a linear relationship between gradient time and retention time the linear solvent strength condition applies to Type II and Type III gradients. Type III gradients, with the fastest re-equilibration time, are preferable for repetitive analyses. Type I gradients are relatively ineffective in making use of the solvent strength of CHAPS, and Types I and II gradients require long equilibration times due to large changes in surface concentration of CHAPS which occur during elution. The presence of CHAPS had a negligible effect on peak shapes of the proteins examined, except for bovine serum albumin which yielded a narrower, less distorted peak in the presence of CHAPS.     

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.     

Use of the surfactant 3-(3-cholamidopropyl)-dimethyl-ammoniopropane sulfonate in hydrophobic interaction chromatography of proteins

Isocratic hydrophobic interaction chromatography of five proteins has been carried out using mobile phases containing the surfactant 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS). Linear relationships were found between log k' and ammonium sulfate concentrations for all the proteins with CHAPS in the submicellar concentration range. The slope of such a plot decreases monotonically as CHAPS concentration is increased. To a first approximation, the effect of CHAPS on protein retention can be explained in terms of a competitive binding model. However, CHAPS does show differential effects on the elution of proteins, substantially altering selectivity. The use of a normalized capacity factor, k'/k'o, proves useful for comparing retention times of different proteins as a function of CHAPS concentration. The magnitudes of k'/k'o were found to be inversely correlated with the slopes of plots of log k' vs. ammonium sulfate concentration in the absence of CHAPS. Adsorption isotherms for CHAPS were determined over the working range of ammonium sulfate. The binding of CHAPS to the SynChropak Propyl stationary phase and its effects on retention were found to be readily reversible. For each protein, plots of k'/k'o vs. surface concentration of CHAPS were superposable for data obtained at different salt concentrations. These findings support a competitive binding model. A simple geometric argument for stationary phase occupancy provides a qualitative explanation for the observed surfactant selectivity.     

Chromatography of beta-glucuronidase from bovine liver. A study of the enzyme binding sites of prepared adsorbents.

beta-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-beta-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that beta-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 M concentration of the salt with p-aminophenyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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。     

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.     

On-column refolding of consensus interferon at high concentration with guanidine-hydrochloride and polyethylene glycol gradients

Dilution refolding of consensus interferon (C-IFN) had a limit on final concentration not exceeding 0.1 mg ml(-1) in order to achieve specific activity of 2.2x10(8) U mg(-1). Addition of polyethylene glycol (PEG) only gave a marginal improvement on the specific activity. Hydrophobic interaction chromatography (HIC) was tried but a simple step-wise elution could not refold the protein. Successful refolding was achieved by gradient elution with the decreasing of guanidine-hydrochloride (guanidine-HCl) concentration. The column was packed with a commercially available HIC medium that was designed for protein separation. Polyethylene glycol was found to possess better effect on the column than in the dilution for promotion of correct refolding, especially in gradient mode. A novel dual-gradient strategy, consisting of decreasing guanidine-HCl concentration and increasing PEG concentration, was developed to enhance the refolding yield. Denatured C-IFN was allowed to adsorb and elute from the HIC column through a gradually changed solution environment. Compared with dilution refolding, the gradient HIC process, in the presence of PEG, gave about 2.6-folds of increase in specific activity, 30% increase in soluble protein recovery. Partial purification was also achieved simultaneously.     

Comparative study on the interaction of recombinant human soluble catechol-O-methyltransferase on some hydrophobic adsorbents

The main scope of this work is the evaluation and potential application of hydrophobic interaction chromatography in the isolation of recombinant human soluble catechol-O-methyltransferase (hSCOMT) from an Escherichia coli cell extract. Therefore, a comparative study on the interaction of recombinant hSCOMT with different hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose), was developed. The four adsorbents were evaluated in terms of selectivity, recovery and fractionation of recombinant hSCOMT from its Escherichia coli-free culture broth. Our data shows that the adjustment of the ionic strength on the mobile phase and the type of hydrophobic ligand are the most useful factors for a complete binding of hSCOMT and a selective fractionation of contaminating proteins. The results of these studies demonstrate that, although epoxy-Sepharose is used as a last resort due to the high salt concentrations needed, hSCOMT bind to the other three resins at low concentrations of ammonium sulfate (< or = 0.6 M) and eluted just by decreasing the ionic strength on the eluent to 0 M, without loss of specific of activity. The stepwise gradient with 0.6, 0.2, 0.075 and 0 M of ammonium sulfate onto a butyl-Sepharose media was found to be the most effective in the isolation of hSCOMT, leading to an enzyme solution with a 3.9-fold increased in specific activity. Since biochemical and structural studies require significant quantities of the enzyme in an active form, the above described approach may give some insight into the optimization and development of new purification strategies of mammalian COMTs.     

Application of a chromatography model with linear gradient elution experimental data to the rapid scale-up in ion-exchange process chromatography of proteins

We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100mL and 2.5L column sizes was performed both by the separation of beta-lactoglobulin A and beta-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method.     

Preparation of poly(N-isopropylacrylamide)-grafted polymer monolith for hydrophobic interaction chromatography of proteins

Poly(N-isopropylacrylamide)-grafted polymer monolith has been achieved using a surface-initiated atom transfer radical polymerization grafting polymerization within the pores of poly(chloromethylstyrene-divinylbenzene) macroporous monolith contained in a 100 mm × 4.6 mm I.D. stainless steel column. The grafted-poly(N-isopropylacrylamide) on the surface of the grafted monolith that was used as chromatographic stationary phase showed a response to the variation of temperatures and/or salt concentrations. This study focus on its salt concentration responsive property and it has been revealed that the hydrophobicity of the grafted monolith can be adjusted by changing salt concentrations in the range of 0.05-2.0 mol/L. A variety of salts including sodium sulfate, ammonium sulfate and sodium chloride exhibited different effects on the alteration of hydrophobicity of the grafted monolith, and the effect of the salts was in the order of sodium sulfate > ammonium sulfate > sodium chloride. Based on this response to salt concentrations, the grafted monolith was applied in hydrophobic interaction chromatography of proteins, and the base-line separation of a six proteins mixture consisting of cytochrome c, myoglobin, ribonuclease A, bovine serum albumin, ovalbumin and thyroglobulin bovine was achieved by a salt gradient elution.