Large scale purification of factor X by hydrophobic chromatography

Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.     

Refolding and purification of interferon-gamma in industry by hydrophobic interaction chromatography

A new technology for renaturation with simultaneous purification of the recombinant human interferon-gamma (rhIFN-gamma) in downstream of biotechnology is presented. The strategies to develop the new technology in industry scale were suggested. Based on chemical equilibrium and molecular interactions, the principle of rhIFN-gamma refolding by HPHIC was described. The kind of stationary and mobile phases were evaluated and found the former to contribute to the rhIFN-gamma refolding more than the latter. The extract containing the rhIFN-gamma in gram scale in 7.0 mol L(-1) guanidine hydrochloride solution of 700 mL was directly pumped into a unit of simultaneous renaturation and purification of proteins (USRPP, 10 x 300 mm i.d.) packed by small particle packings of hydrophobic interaction chromatography and a satisfactory recovery of bioactivity and mass of the rhIFN-gamma was obtained. With flow rate 100 mL min(-1) and a gradient elution by only one step in 4h, the purity and specific bioactivity approach to 95% and 8.7 x 10(7) IU(-1) mg, respectively. To evaluate the goodness of the presented new technology in this study, a usual method with the renaturation by dilution method firstly and then purification with a series of LC in literature was employed to compare with each other. The obtained result in terms of purity, recoveries of mass and bioactivity, cost time as well as expenses, the former is much better than the latter. Comparing the total bioactivity of rhIFN-gamma in the extract before to that after the renaturation by the USRPP, the total bioactivity of rhIFN-gamma increased 62-fold.     

One-step purification of bacterial lipid macroamphiphiles by hydrophobic interaction chromatography

Bacterial lipid macroamphiphiles extracted with phenol/water can be purified in one step by hydrophobic interaction chromatography. Lipids and the major part of protein are separated from macroamphiphiles during phenol/water extraction. Coextracted nucleic acids, polysaccharides, and residual protein are effectively removed by column chromatography on octyl-Sepharose whereby macroamphiphiles are primarily adsorbed and later eluted with a buffered propanol gradient. The procedure is applicable to macroamphiphiles with various lipid structures as was demonstrated using the diacylglycerol-containing lipoglycan of Micrococcus luteus, the lipid A-containing lipopolysaccharide of Salmonella typhimurium, and the diglyceryl tetraether lipoglycans of Thermoplasma acidophilum and Thermoplasma volcanicum. On elution from octyl-Sepharose, separation into molecular species of different compositions was observed with the lipopolysaccharide of S. typhimurium and the lipoglycan of T. volcanicum. It was also shown that, after phenol/water extraction, membrane lipids are completely recoverable from the phenol layer, which makes it possible to isolate lipids along with macroamphiphiles from the same sample of bacteria.     

Native purification of biomolecules with temperature-mediated hydrophobic modulation liquid chromatography

The high-resolution purification of native enzymes is impeded by the limitations in the mobile-phase choices required for conventional hydrophobic separations such as in reverse-phase chromatography. To avoid problems associated with varying the composition of the mobile phase, we developed a stationary phase with a hydrophobicity that can be modulated by slight variations in temperature to bind and elute biomolecules. This chromatographic matrix was tested on nucleotide analogs, amino acids, and protein samples. Visualization of the temperature-dependent hydrophobic interaction with the chromatographic matrix was performed with fluorescence microscopy of CY3-ATP. Amino acids adsorbed to the column according to their known hydrophobicities, confirming the hydrophobic nature of their interaction with the matrix. Biomolecules were separated by modulating the hydrophobicity of the column matrix with slight adjustments to the running temperature between 22 and 37 degrees C without changing the mobile phase. Freedom in the choice of a mobile phase for both the loading and the elution of samples provides great practical advantages by eliminating the need for buffer-exchange steps and allowing more native conditions for purifying delicate enzymes, such as myosin.     

Penicillin acylase purification with the aid of hydrophobic charge induction chromatography

The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.     

Application of hydrophobic interaction displacement chromatography for an industrial protein purification

Recently it has been established that low molecular weight displacers can be successfully employed for the purification of proteins in hydrophobic interaction chromatography (HIC) systems. This work investigates the utility of this technique for the purification of an industrial protein mixture. The study involved the separation of a mixture of three protein forms, that differed in the C-terminus, from their aggregate impurities while maintaining the same relative ratio of the three protein forms as in the feed. A batch high-throughput screening (HTS) technique was employed in concert with fluorescence spectroscopy for displacer screening in these HIC systems. This methodology was demonstrated to be an effective tool for identifying lead displacer candidates for a particular protein/stationary-phase system. In addition, these results indicate that surfactants can be employed at concentrations above their CMCs as effective displacers. Displacement of the recombinant proteins with PEG-3400 and the surfactant Big Chap was shown to increase the productivity as compared to the existing step-gradient elution process.     

Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.     

Sequence-specific oligonucleotide purification using peptide nucleic acid amphiphiles in hydrophobic interaction chromatography

We present a methodology to perform sequence-specific separations of oligonucleotides using peptide nucleic acids covalently linked to alkane chains, or "PNA amphiphiles (PNAAs)". The PNAA/DNA duplexes are discriminated from unbound DNA using hydrophobic interaction chromatography on a phenyl-substituted Sepharose column. Nearly quantitative recovery is achieved at concentrations of 50 microM after incubation of oligomers with a stoichiometric amount of PNAA for 1 min or so. The method exhibits high sequence specificity, selectivity, and resolution when applied to mixtures of various oligomers up to 60 base pairs in length.     

Scale-up of hydrophobic interaction chromatography for purification of antitumor antibiotic SN-07

Scale-up of hydrophobic interaction chromatography for purification of SN-07 was studied. The height equivalent to a theoretical plate of the adsorbent gel (Sepabeads FP-PH 12) was kept constant for various superficial liquid velocities and column diameters. The efficiency of purification was also constant for the Sepabeads FP-PH 12 columns ranging from 16 mm to 113 mm in inner diameter. Effects of the ligand concentration on the adsorption capacity were also studied, and the adsorption capacity increased with an increase in the ligand concentration. These effects resulted in decreasing the required amount of salt to purify SN-07.     

Use of ethanol-eluted hydrophobic interaction chromatography in the purification of serum amyloid A.

A two-step procedure for the purification of the acute-phase reactant serum amyloid A from serum is described. A hydrophobic interaction chromatography medium, octyl-Sepharose CL4B, eluted with increasing concentrations of EtOH was used as the first step in the purification. The concentrate from this step was applied to a gel filtration column of Sephacryl S-200 and eluted with 10% formic acid. The overall recovery of purified serum amyloid A from serum was 56%. This represents the first time that serum amyloid A has been purified without the use of high concentrations of guanidine or urea. The method presented could easily be scaled up to allow the purification of large quantities of serum amyloid A or readily adapted to the purification of other serum apolipoproteins.     

Specific purification of glyceraldehyde-3-phosphate dehydrogenase by hydrophobic chromatography on immobilized colchicine

Hydrophobic column chromatography of bovine brain extracts (40-80% ammonium sulfate fraction) on immobilized colchicine resulted in the selective elution of one major protein with decreasing ionic strength of medium. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.1) on the basis of its biochemical properties, N-terminal amino-acid sequence and enzymatic activity. The present method enabled GAPDH to be isolated with a high recovery (80%; 184 mg/kg brain) and could be of potential use for the purification of GAPDH from various tissues.     

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.     

A simple purification of calmodulin by reversed phase chromatography with hydrophobic polymer 3520

A new adsorption chromatography procedure for the purification of calmodulin from bovine brain was developed using polymeric adsorbent 3520. Calmodulin was first isolated by DEAE-Cellulose column chromatography and further purified to apparent homogeneity following elution with 50% ethanol from the adsorbent column. Polyacrylamide gel electrophoresis showed one band either in the presence of Ca2+ or EGTA. The polymeric adsorbent 3520 is a non-polar polymer lacking exchangeable groups. The selective adsorption of calmodulin is based on hydrophobic interaction within the matrix, and is Ca2+ independent. Neither high salt (0.5 M NaC1) nor EGTA (5 mM) was able to elute the CaM from the adsorption column whereas ethanol (50%) eluted it completely. This method is simple to use and it provides highly purified calmodulin with high yield.     

Scale-up of hydrophobic interaction chromatography for the purification of a DNA vaccine against rabies

The purification of a DNA vaccine against rabies by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatised with 1,4-butanediol diglycidyl ether was scaled up 60 times. The purification profile was not affected by increased loadings (up to 15 mg DNA) and a product with a consistent quality was obtained. Fourteen mg of plasmid with an HPLC purity of 100% were obtained in one run, corresponding to a 95% yield.     

Development of a purification procedure for the placental protein 14 involving metal-chelate affinity chromatography and hydrophobic interaction chromatography

Placental protein 14 was isolated from the biological material of patients undergoing legal abortions. The major part of ballast protein was removed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose. Albumin was separated by chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography on Nickel-Chelate Sepharose and hydrophobic interaction chromatography on Phenyl-Sepharose and Octyl-Sepharose. The protein was not exposed to denaturing agents or extreme pH.     

Application of hydrophobic chromatography to the large scale purification of Desulfovibrio desulfuricans cytochrome C3

Hydrophobic chromatography on Phenyl-Sepharose was employed for large scale purification of cytochrome c3 from Desulfovibrio desulfuricans. This chromatographic procedure minimizes operational volumes and eliminates the lengthy dialysis ordinarily used to remove large amounts of salts. Pure cytochrome c3 was obtained after one additional purification by ion-exchange chromatography.     

Simple procedure for purification of diphtheria toxin and separation of its fragments by hydrophobic chromatography

Diphtheria toxin and fragment B bind to hydrocarbon-coated agaroses. Fragment A of the toxin is not adsorbed to such resins. Using Seph-C₄, the toxin and fragment B can be eluted from the column after adsorption by increasing the ionic strength of the eluent. The toxin is also eluted from the Seph-C₆ column, but fragment B is eluted only in the denatured form. Purification of the toxin can be achieved simply by passing the growth medium supernatant through a small size Seph-C₆ column and eluting the toxin by 0.1 m NaCl. The fragments of diphtheria toxin obtained after mild trypsin treatment can be separated purely on a Seph-C₄ column. The hydrophobic chromatography system may thus serve as a tool for purification of the toxin and its fragments: it may also be useful in large-scale preparations.     

Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography

A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio A₂₇₈/A₅₉₇ of 1.10±0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - Tris Tris(hydroxymethyl)aminomethane - FSCE free solution capillary electrophoresis     

High-performance hydrophobic interaction chromatography: purification of rat liver carbamoylphosphate synthetase I and ornithine transcarbamoylase

The applicability of high-performance hydrophobic interaction chromatography using newly developed silica-based ether-bonded phases is demonstrated in the purification of the rat liver enzymes carbamoylphosphate synthetase I and ornithine transcarbamoylase from crude mitochondrial extracts. As a result of the mild adsorption/elution conditions in this high-performance chromatographic mode, the enzymes are recovered in 20 min with 3- to 15-fold increases in specific activity. Since the enzymes are labile and may aggregate in solution, in one case up to Mr 330,000, this rapid purification demonstrates the potential of hydrophobic interaction chromatography in complex biological systems.