Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

A method for large scale purification of turnip peroxidase and its characterization

Purification of peroxidase has been carried out since 1960 from different sources and with different methods. Ion exchange, affinity, hydrophobic, and metal affinity chromatography are known, to our knowledge. The present method, developed in this study, is three-phase partitioning, a novel technique to separate protein directly from a large volume of crude suspension. It has been observed that interfacing phase with a metal makes this technique highly selective. Turnip peroxidase purified with this method has 512 units/mg with 20.3% recovery. The natural proteins containing histidine or cystine are often purified by immobilized metal affinity chromatography. The purification of turnip peroxidase with the three-phase partitioning technique is based on immobilized metal affinity chromatography and is used for large-scale purification. The present method, described here, would prove its value in purifying an industrially important enzyme on a large scale from a crude suspension. The enzyme purified with this technique showed two bands on SDS- PAGE, which showed a molecular weight of approx. 39KD. Enzyme showed maximum purification with Cu++ metal and had a maximum activity at pH 6.0. The enzyme has an affinity towards hydrogen peroxide as its substrate in the presence of orthodianisidine as a chromogenic substrate. Enzyme activity was enhanced with calcium and magnesium, whereas sodium, potassium, and manganese inhibit the enzyme activity.     

Immobilized metal ion affinity chromatography.

The introduction of immobilized metal ion affinity chromatography, directed toward specific protein side chains, has opened a new dimension in protein purification. This review covers the principles and practice of IMAC that can be performed under very mild, nondenaturing conditions. IMAC is particularly suitable for preparative group fractionation of complex extracts and biofluids, but can also be used in high-performance mode: "HP-IMAC." Single-step purifications of 1000-fold or more may allow isolation of a particular protein from crude extracts on a milligram or gram scale. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography, and the immobilized metal ion ligand complexes are more likely to withstand wear and tear than are antibodies or enzymes. The enormous potential of IMAC and related metal affinity techniques is only in the initial stages of being explored and exploited. Synthesis of IMA adsorbents, and various modes of performing IMAC are discussed and exemplified with selected applications. Advantages and disadvantages are listed. Effective means of counteracting the few undesirable effects that can occur are suggested.     

Immobilized metal ion affinity chromatography: a review on its applications

After 35?years of development, immobilized metal ion affinity chromatography (IMAC) has evolved into a popular protein purification technique. This review starts with a discussion of its mechanism and advantages. It continues with its applications which include the purification of histidine-tagged proteins, natural metal-binding proteins, and antibodies. IMAC used in conjunction with mass spectroscopy for phosphoprotein fractionation and proteomics is also covered. Finally, this review addresses the developments, limitations, and considerations of IMAC in the biopharmaceutical industry.     

Recovery and purification of aprotinin from industrial insulin-processing effluent by immobilized chymotrypsin and negative IMAC chromatographies

Aprotinin, a bovine protease inhibitor currently also produced in recombinant bacteria, yeast, and corn, has valuable applications as a human therapeutic and in tissue culture. The objective of this work was to develop the basis of a large-scale aprotinin purification process centered on immobilized metal ion affinity chromatography (IMAC). This technique uses ligands—metal ions—of a lower cost and higher stability than those traditionally used in affinity chromatography. Since aprotinin does not interact with IMAC ligands, collection is from the nonretained fractions (negative chromatography). Stirred-tank batch IMAC adsorption experiments indicated that one-step aprotinin purification could not be successful. Immobilized chymotrypsin chromatography was then used as a prepurification step, yielding a suitable feed for IMAC (with purification factors as high as 476). IMAC column fed with these prepurified materials produced purified aprotinin in the nonretained fractions with purification factors as high as 952.     

Purification of A-subtype pancreatic cholecystokinin receptor by immunoaffinity chromatography.

So far, no efficient affinity chromatography for CCK receptor purification has been reported that prevented obtention of sequenceable amounts of purified receptor. In this work, 10% of plasma membrane receptor sites were specifically cross-linked with the photoreactive cleavable agonist 125I-ASD-[Thr28, Ahx31]-CCK-25-33, solubilized by NP-40, chromatographied on immobilized wheat germ agglutinin and further immunopurified using anti-CCK antibodies to an overall rate of 3000-3600-fold. Analysis of eluted material demonstrated a protein migrating at Mr 85,000-100,000 and the absence of 35S-labeled impurity. This single and efficient affinity chromatography should provide enough homogeneous receptor protein for microsequence determination and leads to consider immunoaffinity chromatography on immobilized anti-ligand antibodies as a potential tool for purification of membrane receptors.     

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.     

重组蛋白质纯化技术

90年代以来 ,基因重组技术得到很大的发展 ,基因工程产品的分离纯化的成本约占其全部成本的 6 0 %～ 80 %,因此重组蛋白的分离纯化技术越来越重要。着重介绍了扩张柱床吸附层析技术 ,径向膜层析技术 ,灌注层析技术 ,液液萃取技术 ,置换层析技术和金属螯合亲和层析技术近年来进展情况以及它们的优缺点和应用范围。     

Automated large-scale purification of a G protein-coupled receptor for neurotensin

Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.     

Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study

Polyvinyldiene fluoride (PVDF)-based affinity membranes with immobilized copper ions were developed in this study. The resulting membranes were tested for their adsorption properties using a model protein, lysozyme, in batch mode. First, different lengths of diamine were utilized as spacer arms to immobilize the metal ions onto the membranes. It was found that the application of 1,8-diaminooctane as the spacer arm led to the highest adsorption capacity. Moreover, the effects of pH and salt concentration were investigated to distinguish the proportion of specific and nonspecific interactions. A big fraction of lysozyme adsorption capacity for the immobilized metal affinity membranes was considered to come from nonspecific electrostatic interactions, which could be reduced by increasing salt concentration. Lastly, the purification of hepatocyte growth factor (HGF) from insect cell supernatant was performed using the immobilized metal affinity membranes in batch mode. HGF was found in the elution condition using EDTA, indicating the successful purification of HGF.     

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.     

Selective adsorption of apoferritin on immobilized Fe(III): demonstration of Fe(III) binding sites

Immobilized metal ion affinity chromatography has been used to demonstrate and partially characterize Fe(III) binding sites on apoferritin. Binding of Fe(III) to these sites is influenced by pH, but not affected by high ionic strength. These results suggest that both ionic and coordinate covalent interactions are important in the formation of the Fe(III): apoferritin complex. This is, to our knowledge, the first demonstration of direct Fe(III) binding to apoferritin. Other immobilized metal ions, including Zn(II), Ni(II), Cu(II), Cr(III), Co(II), and Tb(III), displayed little or no adsorption of apoferritin. The analytical technique of immobilized metal ion affinity chromatography also shows great promise in the purification of apoferritin, ferritin, and other iron-binding proteins.     

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.     

Recent Progress in the Purification of Adenosine Receptors

Abstract

A₁ adenosine receptors were purified to an apparent homogeneity from rat brain and testicular membranes by a novel affinity chromatography system using xanthine amine congener (XAC) as an immobilized ligand. This affinity chromatography was also useful for the purification of human brain A1 adenosine receptor.     

Protein binding to polymer brush,based on ion-exchange,hydrophobic, and affinity interactions

The major limitations associated with conventional packed bed chromatography for protein separation and purification can be overcome by using adsorptive microporous membranes as chromatographic media. Microporous membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate diffusion limitations. As a result, higher throughput and shorter processing times are possible using these membrane systems. In this paper, we review the current state of development in the area of attaching functionalized polymer brushes onto a microporous membrane to form a novel chromatographic medium for protein separation and purification. The functionalized polymer brushes were appended onto the pore surface of a microporous hollow-fiber membrane uniformly across the membrane thickness by radiation-induced graft polymerization and subsequent chemical modifications. We review various applications of this adsorptive membrane chromatography by focusing on polymer brushes bearing ion-exchange, hydrophobic and affinity groups. Proteins were captured in multilayers by the ion-exchange group-containing polymer brushes due to the formation of a three-dimensional space for protein binding via the electrostatic repulsion of the polymer brushes. In contrast, proteins were captured in a monolayer at most by the polymer brushes containing hydrophobic or affinity ligands. By permeating a protein solution through the pores rimmed by the polymer brushes, an ideal capturing rate of the proteins with a negligible diffusional mass-transfer resistance was achieved by the functionalized polymer brushes, based on ion-exchange, hydrophobic, and affinity interactions.     

Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography

While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.     

包涵体纯化技术

20世纪90年代以来,基因重组技术得到很大的发展,基因工程产品的分离成本约占其全部成本的60%～80%,因此纯化技术越来越重要。着重介绍包涵体纯化技术,包括金属亲和层析,凝胶过滤层析,离子交换层析,疏水层析,双水相萃取技术和反胶团相转移技术近年来的进展情况。     

Protein fractionation using fast flow immobilized metal chelate affinity membranes

A new group-specific affinity membrane using metal chelates as ligands and inorganic glass hollow fiber microfiltration membranes as support matrices is developed and tested. The study focused on developing the optimum activation and coupling procedures to bind the chelating agent (iminodiacetic acid, IDA) to the surface of the microporous glass hollow fiber membrane and testing the resultant affinity membrane. Starting with three different glass surfaces, five modification reactions were evaluated. All the modified "active surfaces" were first tested for their protein adsorptive properties in batch mode with suspended microporous glass grains using model proteins with known binding characteristics with Cu-IDA systems. The metal loading capacities of the surfaces exhibiting favorable fractionation were then measured by atomic absorption spectroscopy.The results were compared with the results obtained with a commercial material used in immobilized metal affinity column chromatography. The protein binding characteristics of the hollow fiber affinity membranes were also evaluated under conditions of convective flow. This was performed by flowing single solute protein solutions through the microporous membrane at different flow rates. These results were then used to estimate the optimum loading and elution times for the process. A mathematical model incorporating radial diffusion was solved using a finite difference discretization method. Comparison between model predictions and experimental results was performed for four different proteins at one flow rate. These results suggested that the kinetics of adsorption was concentration dependent. Finally, the hollow fiber affinity membranes were challenged with two component mixtures to test their ability to fractionate mixed protein solutions. Efficient separation and good purity were obtained.The results presented here represent the development of a new fast flow affinity membrane process-immobilized metal affinity membranes (IMAM). (c) 1994 John Wiley & Sons, Inc.     

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).