Non-chromatographic Method Research Articles

Magnetic aqueous two phase fishing: A hybrid process technology for antibody purification

The potential to combine aqueous two-phase extraction (ATPE) with magnetic separation was here investigated with the aim of developing a selective non-chromatographic method for the purification of antibodies from cell culture supernatants. Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and dextran were supplemented with several surface modified magnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions. The binding capacity of gum arabic coated particles modified with aminophenyl boronic acid (GA-APBA-MP) was were found to be excellent in combination with the ATPS system, yielding high yields of antibody recovery (92%) and purity (98%) from cell culture supernatants. The presence of MPs in the ATPS was found to speed up phase separation (from 40 to 25min), to consume a lower amount of MPs (half of the amount needed in magnetic fishing) and to increase the yield and purity of a mAb purified from a cell culture supernatant, when compared with ATPE or magnetic fishing processes alone.

A Rapid, Convient and Diversified-Multicomponent Synthesis and Characterization of Pyrano[2,3-<i>b</i>]Indoles

A series of pyrano[2,3-b]indoles, was efficiently synthesized via one-pot, multi component reaction (MCRs) of 1,3-bifunctional synthon(malononitrile/ethylcyano acetate), aromatic aldehydes and oxiindole in the presence of various basic catalyst. The key advantages of this process are high yields, shorter reaction times, easy work-up, and purification of products by non-chromatographic method.

A Rapid, Convient and Diversified-Multicomponent Synthesis and Characterization of Pyrano[2,3-<i>b</i>]Indoles

A series of pyrano[2,3-b]indoles, was efficiently synthesized via one-pot, multi component reaction (MCRs) of 1,3-bifunctional synthon(malononitrile/ethylcyano acetate), aromatic aldehydes and oxiindole in the presence of various basic catalyst. The key advantages of this process are high yields, shorter reaction times, easy work-up, and purification of products by non-chromatographic method.

InCl3-catalyzed green synthesis of 1H-pyrazolo[1,2-b]phthalazine-5,10-diones under solvent-free conditions

Abstract A simple, efficient, and green practical approach to 1H-pyrazolo[1,2-b]phthalazine-5,10-diones from phthalhydrazide, aldehydes, and malononitrile/ethyl cyanoacetate has been developed that uses inexpensive and readily available InCl3 as a catalyst in solvent-free one-pot three-component cyclo condensation reaction. This method should provide high yields, shorter reaction time, easy work-up, purification of products by nonchromatographic method, and cleaner reaction. It is a new strategy for N-fused heterocycles synthesis, which haswider application in organic and medicinal chemistry.

IgG PURIFICATION USING AFFINITY FILTRATION WITH SULFAMETHAZINE-AFFINITY CARRIERS

Immunoglobulin G (IgG) antibodies are used extensively for analytical, diagnostic, and therapeutic applications. However, there are some disadvantages to purify IgG antibodies by protein A and G affinity chromatography. Therefore, it is necessary to find an effective alternative and nonchromatographic method to purify IgG. Dextran microparticles were activated and coupled with sulfamethazine to form sulfamethazine-affinity carriers. Then the carriers were used to purify IgG by affinity filtration. Quantitative and qualitative determination proved that sulfamethazine would successfully bond to the surface of dextran microparticles with a density of 85.5 μmol/g (wet). Affinity carriers were proved to withstand high shear force and reveal rare sulfamethazine leakage under filtration conditions between pH 3 to 11. The maximum IgG-binding capacity of affinity carriers was 8.03 mg IgG/g (wet). The affinity filtration process obtained a recovery yield above 80% and purity above 90%. Thus, this work involved in both the advantages of membrane filtration and affinity purification. The results, for the first time, proved that it is possible to use the small ligand sulfamethazine for affinity filtration of IgG. It is an attractive alternative to conventional protein A or G affinity chromatography.

Role of smart polymers in protein purification and refolding

Affinity precipitation is a non-chromatographic method which is useful for purification and refolding of proteins. Quite often, a stimuli-sensitive polymer can be identified which selectively binds to the desired protein. For separation, the protein can be recovered from the precipitate of the protein-smart polymer complex. In case of a refolding experiment, binding of the solubilized protein (in its denatured form) with the polymer leads to the refolding of the protein.

Highly efficient solvent-free synthesis of pyranopyrazoles by a Brønsted-acidic ionic liquid as a green and reusable catalyst

A simple, green and efficient protocol for synthesis of dihydropyrano[2,3-c]pyrazole derivatives is developed by a four component reaction of various benzaldehydes, ethyl acetoacetate, hydrazine hydrate and malononitrile in the presence of 3-methyl-1-(4-sulphonic acid)butylimidazolium hydrogen sulphate [(CH2)4SO3HMIM][HSO4], an acidic ionic liquid and as a catalyst, under solvent-free conditions. The key advantages of this process are high yields, shorter reaction times, easy work-up, purification of products by non-chromatographic method and the reusability of the catalyst. A simple and efficient protocol for synthesis of dihydropyrano[2,3-c]pyrazole derivatives is developed by using [(CH2)4SO3HMIM][HSO4], as a catalyst.

Nonchromatographic method for acid sphingomyelinase in WBC lysate using modified Dole solvent

Correspondence: Mohammad Zouheir Habbal Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, PO Box 11-0236, Riad El Solh 110-72020, Beirut, Lebanon Tel +96 11 350 000 ext 5220 Fax +96 11 370 845 Email mh03@aub.edu.lb Purpose: To use a modified fluorometric method to measure white blood cell acid sphingomyelinase activity. Methods: White blood cell lysates were prepared and used as a source of the enzyme. Two tubes were used for each assay, the second as a blank. In each, N-(NBD-Aminolauroyl)sphingomyelin dissolved in chloroform-methanol (2:1) was added and the organic solvent was removed by nitrogen gas. Acetate buffer, 1% Triton X100 solution, and sonicated protein (the reaction tube only) were added to the residue. The mixture in each was then incubated at 37°C for 2 hours, which was followed by the addition of buffer, Dole solvent, heptane, and sodium chloride solution. The sonicated protein was added to the blank tube and NBD-ceramide was extracted by vortex for 5 minutes and brief centrifugation at 3000 rpm. The intensity of fluorescence of the upper phase was determined in a fluorometer at an excitation wavelength of 465 nm and emission wavelength at 530 nm. Results: In 20 normal patients, the range of enzyme activity was 305–1008 pmol with a mean of 570 pmol. In a proven case of Niemann–Pick disease type A by molecular gene analysis, enzyme activity was undetectable. Conclusion: The modified method is convenient in a biomedical genetics laboratory where a request for sphingomyelinase is very infrequent.

A novel polymeric catalyst for the one-pot synthesis of 2,4,5-triaryl-1H-imidazoles

An efficient synthesis of 2,4,5-trisubstituted imidazoles is achieved by three component cyclocondensation of benzil or benzoin, aldehyde and ammonium acetate by using novel polymeric catalyst [poly(AMPS-co-AA)] under solvent-free conditions. The key advantages of this process are high yields, shorter reaction times, easy work-up, purification of products by non-chromatographic method and the reusability of the catalyst. In this work, we report an efficient synthesis of 2,4,5-trisubstituted imidazoles by three component cyclocondensation of benzil or benzoin, aldehyde and ammonium acetate by using novel polymeric catalyst [poly(AMPS-co-AA)] under solvent-free conditions.

A highly efficient and reusable heterogeneous catalyst for the one-pot synthesis of tetrasubstituted imidazoles

An efficient synthesis of 1,2,4,5-tetrasubstituted imidazoles is achieved by four component cyclocondensation of benzil or benzoin, aniline or benzyl amine, aldehyde and ammonium acetate by using novel polymeric catalyst [poly(AMPS-co-AA)] under solvent-free conditions. The key advantages of this process are high yields, shorter reaction times, easy work-up, purification of products by non-chromatographic method and the reusability of the catalyst.

Simultaneously determination of methyl and inorganic mercury in fish species by cold vapour generation atomic absorption spectrometry

A simple and rapid non-chromatographic method was developed to determine methylmercury (MeHg) and inorganic mercury (iHg) levels in muscles tissues of 10 freshwater fish species. The MeHg and iHg were determined by cold vapour atomic absorption spectrometry after alkaline wet digestion of samples. The digested samples were reduced sequentially with stannous chloride and sodium tetrahydroborate for iHg and MeHg, respectively. Parameters such as carrier gas flow rate (argon), volume of oxidizing and potassium persulphate solutions were investigated in detail. The accuracy of the technique was evaluated by using certified reference material (DORM-2) and spiking the both Hg species in muscles tissue of a fish. The limits of detection were 0.117 and 0.133 μg kg(-1) for MeHg and iHg, respectively. The concentrations of MeHg and iHg in muscles tissues of ten fish species were found in the range of (28.4-56.3) and (3.01-8.11) μg kg(-1), respectively.

Sulfuric Acid Immobilized on Silica Gel as Highly Efficient and Heterogeneous Catalyst for the One-Pot Synthesis of 2,4,5-Triaryl-1H-imidazoles

Application of sulfuric acid immobilized on silica gel as an efficient and benign catalyst has been explored in the syn-thesis of 2,4,5-Triaryl-1H-imidazoles via condensation reaction of benzil or benzoin, aldehyde and ammonium acetate. The key advantages of this process are high yields, cost effectiveness of catalyst, easy work-up, purification of products by non-chromatographic method and the reusability of the H2SO4.SiO2 catalyst.

A chemoselective Ugi-type reaction in water using TMSCN as a functional isonitrile equivalent: generation of heteroaromatic molecular diversity

KF-mediated nucleophilic activation of TMSCN as a functional isonitrile equivalent establishes an efficient and chemoselective Ugi-type multicomponent reaction of a heterocyclic amidine and aldehyde with TMSCN in water. In this approach, the use of isocyanide is circumvented, known competing reactions are virtually eliminated, pure products are obtained by a non-chromatographic method, and therapeutically relevant and diverse N-fused 3-aminoimidazoles can be prepared from a wide variety of aldehydes and heterocyclic amidines. This reaction coupling with cascade cyclization provides various privileged tetracyclic heteroaromatic scaffolds.

A non-chromatographic method for the separation of highly pure naphthalene crystals from pyrolysis fuel oil

An innovative approach towards the extraction of highly pure naphthalene crystals was developed using efficient and inexpensive non-chromatographic solvent extraction method from pyrolysis fuel oil (PFO). Self-crystallization of naphthalene was established during the cooling process of extracted liquor. Naphthalene crystals were recrystallized with ethanol and fully characterized by various analytical techniques such as 1H and 13C NMR, FT-IR, TGA, GCMS etc. In general, the solvent extraction and self-crystallization techniques were capable of producing accurate data for native naphthalene from PFO.

An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases

Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.

Silica-Gel–Catalyzed Efficient Synthesis of Quinoxaline Derivatives Under Solvent-Free Conditions

Quinoxaline derivatives have been prepared in good to excellent yields using silica gel as catalyst from various 1,2-diketones and 1,2-diamines under solvent-free conditions for the first time. The advantages of this method are short reaction time, reaction simplicity, convenient workup procedure, and purification of compounds by a nonchromatographic method.

Automatic optosensing device based on photo-induced fluorescence for determination of piceid in cocoa-containing products

Piceid (3,4',5-trihydroxystilbene-3-β-D: -glucoside) is a stilbene which occurs naturally in various families of plants and has been shown to protect lipoproteins from oxidative damage and to have cancer chemopreventive activity. This paper deals with the determination of piceid in cocoa-containing products by using photo-induced fluorescence and the aid of a multicommutated continuous-flow assembly which was provided with an on-line photoreactor. A strongly fluorescent photoproduct is generated from piceid when it is irradiated under UV light for 30s, which is retained on Sephadex QAE A-25 and directly monitored on this active solid support at 257/382nm (λ (exc)/λ (em), respectively). The pre-concentration of the photoproduct of piceid on the solid support greatly improves both sensitivity and selectivity. The influence of different experimental parameters, both chemical (pH, ionic strength) and hydrodynamic (irradiation time, flow rate, photoreactor length, sampling time), was tested. The sample pre-treatment included delipidation with toluene and cyclohexane, stilbene extraction with ethanol/water (80:20, v/v) and clean-up by solid-phase extraction on C(18) cartridges and methanol/water (40:20, v/v) as eluting solution. This procedure allowed the elimination of the aglycon of piceid, resveratrol and other potential interfering species and a recovery of about a 90% piceid. The method was applied to the analysis of piceid in cocoa powder, dark chocolate and milk chocolate. The quantification limits were 1.4, 1.1 and 0.09mgkg(-1), respectively. Relative standard deviations ranged from 1.8% to 3.1%. This is the first reported non-chromatographic method for determination of piceid in these foods.

Non-chromatographic screening method for the determination of mercury species. Application to the monitoring of mercury levels in Antarctic samples

A simple non-chromatographic method for the determination of mercury (Hg 2+), methylmercury (MeHg +), dimethylmercury (Me 2Hg), and phenylmercury (PhHg +) employing atomic fluorescence spectrometry (AFS) as detection technique was developed. Mercury species showed a particular behavior in the presence of several reagents. In a first stage SnCl 2 was employed for Hg 2+ determination; in a second step, [Hg 2+ + PhHg +] concentration was determined using SnCl 2 and UV radiation. MeHg + decomposition was prevented adding 2-mercaptoethanol. In a third stage, [Hg 2+ + PhHg + + MeHg +] concentration was determined using K 2S 2O 8. Finally, the four species were determined employing NaBH 4. Reagents concentration and flow rates were optimized. The extraction technique of mercury species involved the use of 2-mercaptoethanol as ion-pair reagent. The limits of detection for Hg 2+, PhHg +, MeHg +, and Me 2Hg were 1, 40, 68, and 99 ng L −1 with a relative standard deviation of 1.5, 3.1, 4.7 and 5.8%, respectively. Calibration curve was linear with a correlation factor equal to 0.9995. The method was successfully applied to the determination of the mercury species in two Antarctic materials: IRMM 813 ( Adamussium colbecki) and MURST-ISS-A2 ( Antarctic Krill).

Purification of Escherichia coli RNA polymerase using a self‐cleaving elastin‐like polypeptide tag

A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP alpha subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the beta, beta', and omega subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.

A Non-Chromatographic Method for the Purification of a Bivalently Active Monoclonal IgG Antibody from Biological Fluids

This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4-dinitrophenyl IgG: IgG(DNP); and mouse antidigoxin IgG: IgG(Dgn)) from ascites fluid. This procedure (for IgG(DNP)) has three steps: (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation of cyclic complexes of IgG(DNP) by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; (iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid. This method is applicable to antibodies with a wide range of monovalent binding constants (0.1 microM to 0.1 nM). The multivalent ligands we used (derivatives of DNP and digoxin) isolated IgG(DNP) and IgG(Dgn) from ascites fluid in yields of >80% and with >95% purity. This technique has two advantages over conventional chromatographic methods for purifying antibodies: (i) it is selective for antibodies with two active Fab binding sites (both sites are required to form the cyclic complexes) over antibodies with one or zero active Fab binding sites; (ii) it does not require chromatographic separation. It has the disadvantage that the structure of the hapten must be compatible with the synthesis of bi- and/or trivalent analogues.