Anion Exchange Groups Research Articles

Superporous agarose anion exchangers for plasmid isolation

Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 μmol/ml) showed a plasmid binding of 3–4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 μmol/ml) and showed a correspondingly lower plasmid binding capacity (1–2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70–100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45–75 μm diameter beads; 4 μm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one without enzymatic digestion of RNA. The strategy without RNase gave high plasmid recovery, quantitative removal of protein and a 70% reduction in RNA.

Anion-exchange chromatography using short monolithic columns as a complementary technique for human serum albumin depletion prior to human plasma proteome analysis

In order to enable the detection of low abundance proteins from human plasma, it is necessary to remove high abundance proteins. Among them, human serum albumin and immunoglobulin G represent more than 75% of all such proteins. In this paper, the characterization of short monolithic columns was performed followed by the optimization of a multidimensional approach, known as conjoint liquid chromatography, to deplete human serum albumin and immunoglobulin G from a human plasma sample. Two different chromatographic modes were used: ion-exchange chromatography and affinity chromatography. A monolithic stationary phase (convective interaction media disk) bearing strong anion-exchange groups and another immobilized with protein G were placed in series into one housing. The optimal binding conditions were found that removed a majority of human serum albumin and immunoglobulin G from the human plasma sample. This method was compared to the depletion using a combination of pseudo-affinity and affinity columns. The results of the human serum albumin and immunoglobulin G depletion were confirmed by 2D electrophoresis. It has been shown that anion-exchange and affinity chromatography using convective interaction media monolithic columns can represent an efficient complementary technique for human serum albumin and immunoglobulin G removal from human plasma.

Esterification of lauric acid using lipase immobilized in the micropores of a hollow-fiber membrane

A porous anion-exchange hollow-fiber membrane was prepared by radiation-induced graft polymerization and chemical modification to immobilize lipase for enzymatic reaction in an organic solvent. The amount of anion-exchange group introduced to the porous hollow-fiber membrane was 2.5 mol/kgfiber. A lipase solution was allowed to permeate through the porous anion-exchange hollow-fiber membrane, and lipase molecules that adsorbed onto the grafted polymer brush were cross-linked with glutaraldehyde. The lipase was immobilized at a density of 0.14 kglipase/kgfiber, which was equivalent to a degree of multilayer binding of 20. Esterification was carried out by passing a solution of lauric acid and benzyl alcohol in anhydrous issoctane through the lipase-immobilized membrane, and lipase activity was determined. A reaction percentage of 50% was achieved at space velocity 68 h−1. The maximum immobilized lipase and native lipase activities were 8.9 and 0.38 mol/(h·kglipase), respectively. Thus, the activity of the immobilized lipase was 23.4 times higher than that of the native lipase.

Ion Exchange Properties of the Root Cell Walls Isolated from the Halophyte Plants (Suaeda altissima L.) Grown Under Conditions of Different Salinity

Ion exchange properties of root cell walls of 50- to 60-day-old plants of Suaeda altissima L., grown in nutrient medium in the presence of different NaCl concentration (0.3, 250, 750 mM) were investigated. For all growth conditions, the ion exchange capacity of the cell walls was estimated at various pH values and at different salt concentrations. The total amount of cation and anion exchange groups in the cell walls was determined. It is shown that four ionogenic groups in the polymeric structure of the halophyte cell walls are always observed independently of growth conditions: three are cation exchangeable, and one exchanges anions. The amount of the functional groups of each type is estimated, and the corresponding values of pK a j are calculated. It is shown that acidic properties are enhanced for all types of cation exchangeable groups by increasing electrolyte concentration (NaCl) in the nutrient solution. At the level of ‘optimal salinity’ (250 mM NaCl) the amount of polygalacturonic acid groups in cell walls is approx. 1.5 times greater than for the plants grown at either low (0.3 mM NaCl) or high salinity (750 mM NaCl). Ion exchange capacities of the root cell walls of the halophytes grown in different salt concentration are discussed in terms of S. altissima adaptation to salt stress.

On-Probe Sample Preparation without Washes for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using an Anion Exchange Medium

When the mass spectra of biological samples (proteins, peptides, and so on) are obtained routinely by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a serious problem is the reduction of the ionization efficiency by impurities, such as buffer salts and detergents. We focused our attention on devising a method to maintain the ionization efficiency of protein samples, even in the presence of sodium dodecyl sulfate (SDS), without any extra purification step. Although no protein ion peaks are observed in the presence of 2.5% SDS with the usual methods, the addition of a granular anion exchange silica gel to the matrix solution allowed the protein ion peaks to be obtained with an excellent signal-to-noise ratio. Together with other supporting experiments, we suggest that the positively charged surface (the basic environment derived from the anion exchange groups) and the roughness of the particles were important for good ionization in the presence of a high SDS concentration. For a very uneven surface, the SDS might be absorbed into the particle interiors during the process of cocrystallization with the matrix and analytes, which is known as the molecular sieve effect, and the SDS concentration in the surface crystalline film might be reduced. As a result, we developed an on-probe sample preparation method without washes for MALDI, using a strong anion exchange silica gel. This method is applicable even in the presence of 2.5% SDS, and is not only very simple but also inexpensive, because it can be used with the standard MALDI target plates.

174. Process Development of a Scaleable Purification Protocol for the Production of Reolysin, an Oncolytic, Reovirus-Based Therapeutic

Reovirus is an enterovirus that specifically infects cells with an activated ras-pathway. As the activation of the ras-pathway is a common denominator in a wide variety of cancer types, reovirus serotype 3 has been shown to cause regression in a variety of tumors in cell line, animal models as well as in the clinic. To meet the demands of producing infectious virus at large scale for clinical phase II/III trials a filtration and chromatography based approach was developed. We report here the development of a fully scaleable process for the purification of active virus from cell culture. The three step process consists of ultrafiltration, anion exchange purification and group separation into formulation buffer. The process was scaled-up to a 20 liter bioreactor scale and transferred to production in a GMP facility. The overall recovery of the process is >50% and the final purity matches material produced by two sequential cesium chloride centrifugation steps.

Local structures of ions at ion-exchange resin/solution interface

The local structures of Cl − and Br − in anion-exchange resins have been studied by X-ray absorption fine structure (XAFS), and separation selectivity is discussed on the basis of results. When two different anion-exchange resins having trimethylammonium and dimethylammonium groups as anion-exchange groups are employed for ion-exchange experiments, slightly higher Br − selectivity has been obtained with the former. XAFS has indicated that the average hydration numbers for a given anion is not affected by the structure of the ion-exchange group, but that the extent of ion-association between the anion and the ion-exchange groups depends on the type of the ion-exchange group. Shorter interaction distance (and in turn stronger ion-association) has been confirmed for the dimethylammonium-type resin, and is consistent with lower Br − selectivity of this resin.

Preparation of organic–inorganic composite anion-exchange membranes via aqueous dispersion polymerization and their characterization

Organic–inorganic composite membranes based on poly(vinyl alcohol)/SiO 2 were prepared via an aqueous dispersion polymerization route and anion-exchange groups were introduced in the membrane matrix by the chemical grafting of 4-vinylpyridine with the desired content. These membranes were extensively characterized for their surface morphology, thermal stability, water content, and surface-charge properties using SEM, TEM, FTIR, TGA, water uptake, and ion-exchange capacity measurements. Counterion transport numbers across these membranes were estimated from membrane potential data. Membrane conductance measurements were also performed and these data were used for the estimation of values of counterion diffusion coefficients in the membrane phase. Physicochemical and electrochemical properties of these membranes and equivalent pore radius (estimated from electroosmotic flux measurements) were found to be highly dependent on the 4-vinylpyridine (4-VP) content in the membrane phase. It was also observed that for better selectivity and membrane conductivity of anion-exchange membranes complete optimization of the loading of 4-VP in the membrane phase is necessary. Furthermore, among these, membrane with 25% loading with 4-VP exhibited very good selectivity, water content, and ion-exchange capacity along with moderate membrane conductivity, which may be used for their application in electro-driven separation at elevated temperatures or for other electrochemical processes.

Separation of biomolecules by μ-high-performance anion-exchange chromatography using a tentacle-like stationary phase

A variety of biomolecules and their variants, which have previously been problematic to separate, have been analysed using a novel anion-exchange resin based on a non-porous polystyrene polymeric support with a hydrophilic coating and grafted tentacular quaternary ammonium functional groups. The hydrophilic coating results in minimal interaction between the support and the biomolecule, while the highly flexible tentacular-like anion-exchange groups increase the ionic interaction potential and act as an umbrella to hold the proteins away from the surface. Because of the removal of silanophilic interactions, minimisation of hydrophobic interactions, and the highly flexible nature of the tentacle-like ion exchangers, ionic interactions can therefore dominate the separation. As such this phase is highly suited to the separation of highly charged biomolecules and their variants. This polymeric strong anion-exchange (SAX) support was packed into a fused-silica capillary column and, using a salt concentration gradient, various recombinant proteins were analysed by micro-HPLC resulting in baseline resolution.

Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface

We have prepared a new type of anion exchanger, which effectively discriminates between RNA and plasmid DNA. The material is based on a Sephacryl S-500 HR matrix provided with quartenary amine anion-exchange groups. A distinguishing feature of the beads is that a thin (2–3 μm) outer layer of the beads lacks ion-exchange groups. In the synthesis of these beads the vinyl groups in the outer layer of vinylalkyl substituted Sephacryl S-500 HR beads are reacted with bromine. The resulting layer of bromoalkyl groups are hydrolysed, creating an inert outer layer of hydroxyalkyl groups. Finally, bromination and trimethylamine reactions of the inner vinyl groups provide the beads with a core of cationic groups. Large plasmid molecules will not bind to such beads since they are too large to enter the pores and therefore cannot come into contact with the charged matrix in the inner parts of the beads. RNA and protein molecules present in a cleared lysate, on the other hand, readily enter the pores and become adsorbed. A two-column strategy was developed for plasmid purification (recombinant pBluescript, 5.9 kilo base pairs, kbp). The first column was packed with the restricted access anion-exchanger beads (lid beads) and the second column with normal ion-exchange material (same ligand density as the lid beads). Diluted (3×), cleared lysate was pumped through the tandem columns. The first column was subsequently disconnected from the system and the purified plasmid adsorbed on the second column was eluted in a concentrated form (6×) and with 89% recovery. The two-column procedure removed 99.5% of the RNA and 96% of the proteins.

Radioactive iodine and chloride transport across a paper membrane bearing trimethylhydroxypropylammonium anion exchange groups

The separation performance of radioactive iodine from radioactive waste is examined using the paper membrane made of long length pulp fiber bearing trimethylhydroxypropylammonium anion exchange group. Prepared paper membrane is sturdy and high of ion-exchange capacity as the introduction of ion exchange group into the paper membrane with high density and well-bonding between pulp fibers are achieved. Ion transport across the paper membrane is evaluated with electroconductive membrane permeability derived from transmembrane potential and membrane conductance data, and diffusional membrane permeability derived from transmembrane potential and ionic flux in Na 125 I or Na 36 Cl single electrolyte concentration-cell system. The ratio of electroconductive membrane permeability of 125 I to that of 36 Cl reaches 6.2, while the ratio of diffusional membrane permeability of 125 I to that of 36 Cl is almost unity. The novel prepared paper membrane exhibits the 125 I permselectivity by solution/membrane distribution process due to electrostatic effect.

A Simple Collection and Separation Method Using a Small Anion-Exchange Column for the Determination of Sulfide in Sediments

A simple collection and separation of sulfide in bottom sediments using a small anion-exchange column followed by its spectrophotometric determination has been developed. Hydrogen sulfide, which was generated from an acidified suspension containing sulfide-bearing sediment, was collected on a small column packed with OH-type anion-exchange group (quaternary amine) bonded silica gel (OH-SP) by bubbling nitrogen gas through the solutions. The sulfide adsorbed on the OH-SP was eluted with a potassium nitrate solution, and then it was spectrophotometrically determined by the methylene blue method. The established method was successfully applied to bottom sediment samples taken from brackish Lake Nakaumi.

Determination of nitrate and nitrite by high-performance liquid chromatography in human plasma

A new, accurate, fast and simple method has been implemented by which nitrite and nitrate ions, as stable forms of nitric oxide production were studied. A study of these two ions was carried out by a sensitive and accurate HPLC method with two detectors. The most important advantages of the reported method are: short time of analysis, minimal sample pre-treatment, long life of the analytical column and stable eluent solution. The photodiode array UV–Vis detector detected nitrite and nitrate ions at an absorbance of 212 nm. Much more sensitive electrochemical detection with a WE (glassy carbon) electrode was used for the detection of nitrite ions. An analytical chromatographic column was formed by a sorbent, containing strong base anion-exchange groups bound in Cl − form in the hydrophilic hydroxyethyl methacrylate matrix. The anions were analysed in human plasma without deproteinization using 0.02 M sodium perchlorate monohydrate as eluent solution at pH 3.9. At this pH organic substances do not affect the analysis. The retention times for nitrite and nitrate were 3.62 and 3.72 min (by electrochemical detection) and 4.44 min, respectively. The method was linear ( r=0.9992, 0.9998, 0.996) within a 1–100 (nitrate), 1–20 μmol/l (nitrite) concentration range.

Separation of 125I from Radioactive Waste with Anionexchange Membrane Treated with Trimethylhydroxypropylamino Group

The membrane characteristics controlling 125I and 36Cl transport across anion exchange paper membranes are examined using four different ion exchange capacities paper membranes treated with trim ethylhydroxypropylamino group. Nonequilibrium thermodynamic analyses for the paper membrane transport process are carried out based on transmembrane potential, membrane conductance, and ion flux measurements. The electroconductive membrane, permeabilities representing the solution membrane ion distribution due to electrostatic effects, and the diffusional membrane permeabilities, representing the migration speed of ions within the membrane phase, for 125I are higher than those for 36Cl in all cases. In the paper membrane treated with trim ethylhydroxypro-pylamino group, 125I diffusion or 125I solution/membrane distribution process can be improved by increasing the concentration of anion exchange sites in the paper membrane. These findings indicate the potential utility of paper membranes prepared in this way for separating 125I from radioactive waste that has been contaminated with chloride ions. The 125I transport performance of such systems may be improved with the use of other anion-exchange groups.

Transport of 125I and 36Cl across an anion-exchange paper membrane

The membrane characteristics controlling 125I and 36Cl transport across anion-exchange paper membranes are examined using three different paper membranes treated with different anion exchange groups: a trimethylhydroxypropylamino group, a diethylaminoethyl group, and a 50% quaternary diethylaminoethyl group. The membrane permeability for 125I is higher than that for 36Cl in all cases. Treatment with trimethylhydroxypropylamino group improves the 125I diffusion process, and the use of 50% quaternary diethylaminoethyl group improves the 125I solution/membrane distribution process.

High conversion in asymmetric hydrolysis during permeation through enzyme-multilayered porous hollow-fiber membranes.

We describe a novel porous hollow-fiber support for immobilizing aminoacylase in multilayers. Epoxy-group-containing polymer chains were grafted onto a porous hollow-fiber membrane by radiation-induced graft polymerization of glycidyl methacrylate, and subsequently a diethylamino group as an anion-exchange group was introduced into the graft chain. Aminoacylase was adsorbed in multilayers by allowing the amioacylase buffer solution to permeate through the pores across the hollow fiber; the graft chains provided three-dimensional space for the enzymes because of their electrostatic repulsion. The adsorbed enzyme at a degree of multilayer binding of 15 was cross-linked with glutaraldehyde to prevent leakage. An acetyl-DL-methionine solution was allowed to permeate through the pores surrounded by the aminoacylase-immobilized graft chain. Production of L-methionine was observed at a 4.1 mol/h per L of the fiber for a space velocity of 200 h(-1), defined as the flow rate of the effluent penetrating the outside surface of the hollow fiber divided by the membrane volume including the lumen.

Comparison of l-tryptophan binding capacity of BSA captured by a polymer brush with that of BSA adsorbed onto a gel network

A polymer brush containing a diethylamino group as an anion-exchange group was appended onto a polymer substrate by radiation-induced graft polymerization and subsequent chemical modifications. Bovine serum albumin as a chiral ligand for l-tryptophan was bound to the polymer brush at a density ranging from 17 to 150 g BSA/l. For comparison, BSA was adsorbed onto the gel network containing a diethylaminoethyl group. The molar binding ratio of l-tryptophan to BSA on the polymer brush was 1.7-fold higher than that to BSA on the gel network.

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (S i) and the swelling coefficient (K cw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pHi = f (S i), the Gregor's equation was employed. It was shown that the Gregor's model fits well the experimental data. The total number of the cation exchange (S t cat) and the anion exchange (S t an) groups were determined in the root cell walls. The number of the functional group of each type (ΔS j) was estimated, and the corresponding values of pK a j were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferich's equation [pK a j = f (C K+)] were determined. It was found that the swelling of root cell walls changes with pH, C K+ and strongly depends on plant species. Within the experimental pH and C K+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (ΔS j, pK a j, K cw), the equation {pK a j = f (CK+)} and the dependencies {K cw = f (CK+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.

Desalination by electrodialysis with the ion-exchange membrane prepared by radiation-induced graft polymerization

Ion-exchange membranes modified with the triethylamine [–N(CH 2CH 3) 3] and phosphoric acid (–PO 3 H) groups were prepared by radiation-induced grafting of glycidyl methacrylate (GMA) onto the polyolefin nonwavon fabric (PNF) and subsequent chemical modification of poly(GMA) graft chains. The physical and chemical properties of the GMA-grafted PNF and the PNF modified with ion-exchange groups were investigated by SEM, XPS, TGA, and DSC. Furthermore, electrochemical properties such as specific electric resistance, transport number of K +, and desalination were examined. The grafting yield increased with increasing reaction time and reaction temperature. The maximum grafting yield was obtained with 40% (vol.%) monomer concentration in dioxane at 60°C. The content of the cation- and anion-exchange group increased with increasing grafting yield. Electrical resistance of the PNF modified with TEA and –PO 3 H group decreased, while the water uptake (%) increased with increasing ion-exchange group capacities. Transport number of the PNF modified with ion-exchange group were the range of ca. 0.82–0.92. The graft-type ion-exchange membranes prepared by radiation-induced graft copolymerization were successfully applied as separators for electrodialysis.

Determination of plasma methoxyamines.

The determination of plasma methoxyamines is the most informative parameter for the diagnosis of pheochromocytoma. Very good sensitivity and specificity are necessary for this. The measurement of deconjugated (free plus sulfate-conjugated) metanephrines is more useful. Concentrations are 10-fold higher than free metanephrine concentrations. The methodology generally uses a column purification and high performance liquid chromatography with electrochemical detection. We have tested six commercial columns used for the purification step. We chose a rapid and reliable method with a mixture of strong cation-exchange and strong anion-exchange groups bonded onto a silica column. This protocol has been validated with samples of plasma from normal subjects, healthy elderly people, renal failure patients and patients with pheochromocytoma.