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PubMed1988

Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans.

Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity. The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds. In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method. The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E. coli alkaline phosphatase obtained commercially. These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme. Scaling up with larger columns should allow purification of enzymes of a commercial basis.

Alkaline Phosphatase↗
PubMedDate not supplied

Behavior of the inadvertent pH transient formed by a salt gradient in the ion-exchange chromatography of proteins.

The inadvertent pH transient produced when a stepwise change in salt concentration is used as the eluent in ion-exchange chromatography was studied theoretically using a local-equilibrium theory and experimentally using both strong-base and weak-base anion-exchange column packings. The accuracy of the local-equilibrium theory was verified by comparing it to a full numerical solution of the governing partial differential equations obtained using the method of characteristics. The predictions from the local-equilibrium theory were observed to largely agree with experimental results. Detailed comparisons of experimental results and the local-equilibrium theory permitted the observed trends for the pH transients to be interpreted in terms of the physical properties of the column packing and mobile phase. The results of this study are useful for the design of ion-exchange processes using salt gradient elution where it is desired to limit the exposure of eluted proteins to the inadvertent pH transient caused by the salt gradient.

Chromatography, Ion Exchange↗
PubMed1984

The effect of storage conditions on ion exchange and affinity chromatographic assays for glycated hemoglobin.

Experiments were performed to evaluate the effect on glycated hemoglobin determinations when erythrocytes were stored under various conditions. We studied the influence of time, temperature, glucose concentration, and pH on results obtained by three commercially available column chromatographic procedures designed to quantify glycated hemoglobin. An affinity chromatographic procedure which measures total glycated hemoglobins (GHb) was compared with two ion-exchange column methods. One ion-exchange method measures total hemoglobin A1 and the other is purported to measure hemoglobin A1c. Stability studies were performed using specimens from nondiabetics and diabetics, supplemented in vitro with glucose at pH 7.4 and pH 5.0. Results obtained from the A1c and the GHb tests were unchanged over time for samples stored under the described conditions. Erythrocytes were incubated with glucose to produce labile glycated hemoglobin and assayed before and after overnight incubations in saline. These experiments showed that the A1c and GHb assays were unaffected by the presence of labile glycated hemoglobin. Column fractions were analyzed by isoelectric focusing and scanned with a laser densitometer for quantitative assessment of hemoglobin species found in each fraction. These experiments showed that the three column methods measure very different hemoglobin species. Focusing of eluates from the A1 and GHb tests revealed carryover of several hemoglobin species into the measured fractions, while the A1c test was found to be much more specific for HbA1c. Our data suggest that it may be possible to analyze glycated hemoglobins in samples transported to a reference laboratory without special treatment.

Chromatography, Affinity↗
PubMed2002

A direct measurement of the ion-exchange capacity of muscovite mica using a mettler ultramicrobalance.

A direct method is described of measuring the mass of substances dissolved from muscovite mica in water. The mica sheets are weighed by the use of a Mettler ultramicrobalance both before and after the immersion in water. The decrease in weight is regarded as identical to the dissolved mass. An analysis by ICP-AES has confirmed that the dissolution is due to an ion exchange of metal ions, mainly potassium ions. As a result of the measurement of the change in weight of mica sheets with immersion time, it has been found that it takes about 30 min to finish the dissolution. The total mass dissolved from basal plane has been determined irrespective of the difference in fraction of metal ions shared with each of the two mica surfaces obtained by cleavage along the basal plane. The measured values of the total mass differ from specimen to specimen and fall in the range from 1 to 10 mg m(-2). These values of the dissolved mass are comparable to the order of 10 monolayers of potassium ions included in basal plane. The present result suggests that internal metal ions as well as external ions contribute to the ion exchange occurring on the surface of muscovite mica.

Journal Article↗
PubMed1991

Characterization of synthetic macroporous ion-exchange resins in low-pressure cartridges and columns. Evaluation of the performance of Macro-Prep 50 S resin in the purification of anti-Klenow antibodies from goat serum.

Three ion-exchange materials and one hydrophobic-interaction chromatography packing, based on a rigid macroporous polymer with large, relatively uniform pores, have been evaluated for low-pressure liquid chromatography of antibodies. These sorbents have high capacities for both small and large proteins and are mechanically, chemically, and thermally stable. Macro-Prep 50 S. CM and Q ion-exchange materials are strongly acidic, weakly acidic, and strongly basic, respectively. Protein binding and recovery, pressure-flow properties, and chemical and thermal stability were determined for each sorbent. A rapid, two-step method for the purification of anti-Klenow antibodies from goat serum was developed, based on the Macro-Prep 50 S strong-acid cation-exchange material and the Econo-Pac HIC prepacked hydrophobic-interaction cartridge.

Animals↗
PubMed2005

Using computer simulation to assist in the robustness analysis of an ion-exchange chromatography step.

This paper presents a methodology to gain process knowledge and assist in the robustness analysis of an ion-exchange step in a protein purification process using a model-based approach. Factorial experimental design is common practice in industry today to obtain robustness characterization of unit operations with respect to variations in process parameters. This work aims at providing a better insight into what process variations affect quality and to further reduce the experimental work to the regions of process variation that are of most interest. This methodology also greatly increases the ability to predict process performance and promotes process understanding. The model calibration part of the methodology involves three consecutive steps to calibrate a steric mass action (SMA) ion-exchange chromatography model. Firstly, a number of gradient elution experiments are performed. Secondly, experimental breakthrough curves have to be generated for the proteins if the adsorption capacity of the medium for each component is not known. Thirdly, a multi-component loading experiment is performed to calibrate the multi-component effects that cannot be determined from the single-component experiments. The separation process studied in this work is the separation of polyclonal IgG from a mixture containing IgG, myoglobin and BSA. The calibrated model is used to simulate six process variations in a full factorial experiment. The results of the simulations provide information about the importance of the different process variations and the simulations are also used to determine the crucial points for the process parameter variations. The methodology can be used to assist in the robustness analysis normally performed in the pharmaceutical industry today as it is able to predict the impact on process performance resulting from variations in salt concentration, column load, protein concentration and flow rate.

Chromatography, Gel↗
PubMed1985

Removal of hepatitis B virus infectivity from human gamma-globulin prepared by ion-exchange chromatography.

Hepatitis B virus infectivity can be removed from human plasma used to prepare a gamma-globulin product by ion-exchange chromatography with commercially available resins. A pooled human plasma sample free of hepatitis markers was contaminated with 10,000 chimpanzee infectious doses of hepatitis B virus per milliliter from a known infectious inoculum. The contaminated plasma was diluted to yield a weakly positive radioimmunoassay result for HBsAg and then processed over a double-column chromatography system containing anionic ion-exchange resins to yield highly purified gamma-globulin. Two chimpanzees were each inoculated intravenously with this product while a control animal received non-treated material. The 2 experimental animals each received a potential challenge of 3,000 ID and showed no serologic evidence of the disease during 9 months of evaluation. The control animal received 100 ID and developed hepatitis B 25 weeks after the challenge.

Animals↗
PubMed2000

Use of directly coupled ion-exchange liquid chromatography-mass spectrometry and liquid chromatography-nuclear magnetic resonance spectroscopy as a strategy for polar metabolite identification.

Ion-exchange LC-MS and LC-NMR have been successfully used to identify a novel N-acetyl metabolite of a highly polar drug candidate [2-(ethanimidoylamino)ethyl]sulfonyl alanine (GW273629) under development as a therapeutic agent. This has been achieved using a simple HPLC method without the need for complicated and time consuming pre- or post-column derivatisation. Ion-exchange chromatography using simple ionic strength buffer and organic solvent mobile phases, as applied here, should be suitable for the analysis of other charged polar species. Optimisation of the system described could result in the development of a rational generic HPLC approach specifically designed for the characterisation of polar drug molecules and their metabolites.

Animals↗
PubMedDate not supplied

Characterization of polycationic amino acids fusion systems for ion-exchange purification of cyclodextrin glycosyltransferase from recombinant Escherichia coli.

Fusion proteins with charged polycationic amino acid tails were constructed for the purpose of simple ion-exchange purification with high purity. A number of positively charged lysine and arginine tails were fused to the C-terminus of cyclodextrin glycosyltransferase (CGTase) derived from Bacillus macerans and expressed in Escherichia coli. The ionic binding forces provided by the tails allowed the selective recovery of CGTase from recombinant E. coli cell extracts, while CGTase by itself could not bind to the cation exchanger at neutral pH. The type of amino acids used and the length of the tail directly affected the purification factors. Most intracellular proteins of E. coli adsorbed on the cation exchanger could be removed by washing with 400 mM NaCl solution at pH 7.4, suggesting that a fusion partner suitable for purification purpose should be provided with high binding strength and the maintenance of adsorption by washing with NaCl solution. Among the fusion CGTases constructed, the CGTK10ase containing 10 lysine residues provided sufficiently high binding strength to allow purification to its homogeneity through simple ion-exchange chromatography.

Chromatography, Affinity↗
PubMed1986

Ion-exchange and hydrophobic-interaction high-performance liquid chromatography of proteins. A practical study.

A number of commercially available columns for ion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC) have been tested, ranging from very fast columns with low capacity to preparative columns. The experiments represent a selection of column applications likely to occur in a biochemical laboratory. The use of several ion-exchange columns for separation of serum and membrane proteins has been demonstrated. Columns with a silica gel matrix and with a polymer matrix have proved to be of equal value in experiments with water-soluble proteins. In experiments with hydrophobic membrane proteins the choice of the column depends on the protein to be isolated. A combination of a cation-exchange and an anion-exchange column as a tandem as well as a mixed-bed column, were applied for the separation of serum proteins and for the isolation of a 175,000-dalton membrane glycoprotein. One problem of hydrophobic-interaction HPLC is the poor solubility of some proteins, for example serum proteins at high salt concentrations. This difficulty can be overcome by the use of columns with higher hydrophobicity which require lower initial salt concentrations. Less hydrophobic columns have been shown to separate hydrophobic membrane proteins by a combination of salt and detergent gradients.

Animals↗
PubMed2000

Indirect spectrophotometric detection of inorganic anions in ion-exchange capillary electrochromatography.

The application of indirect spectrophotometric detection was investigated for a capillary electrochromatographic system in which an anion-exchange stationary phase (in the form of aminated latex particles) was coated onto the wall of a fused-silica capillary. The study has focused on the choice of the type and concentration of the absorbing coion (probe) added to the background electrolyte and the role of this species in manipulating the ion-exchange contributions to the separation with a view to controlling the selectivity of the separation. Common inorganic anions were used as analytes and nitrate, p-toluenesulfonate, nicotinate, and chromate were investigated as probes. It was found that most of these probes produced only a limited range of separation selectivities when their concentration was varied over the practically accessible range. p-Toluenesulfonate provided the greatest variation in selectivity, but peak distortion due to electromigration dispersion was evident for the faster ions. When variation of the separation selectivity - from predominantly electrophoretic in nature to predominantly ion-exchange in nature - was desired, this was best achieved by varying the type of probe rather than its concentration. For example, the nitrate probe provided predominantly electrophoretic separations with good peak shapes and high efficiencies. A comprehensive list of probes, ranked in order of ion-exchange selectivity coefficients determined by ion chromatography, was compiled and this proved to be a useful tool to assist in the selection of a probe for a desired separation selectivity. The limits of detection for the analytes and probes studied ranged from 20-55 micromol for the chromate system to 230-600 micromol for the nicotinate system, with nitrate and p-toluenesulfonate giving intermediate values.

Anions↗
PubMed1978

Plasma ammonia concentration in dogs using ion-exchange method of analysis.

Laboratory methods for measuring plasma ammonia concentration were reviewed. The ion-exchange method of analysis was evaluated using canine plasma from 15 clinically normal dogs and five dogs with portosystemic shunts. Mean and standard deviation was 40.3 +/- 8.1 microgram NH(3)/dl in normal dogs, and values ranged from 125 to 200 microgram NH(3)/dl in dogs with portosystemic shunts. Handling of blood samples properly for accurate results was found to require pre-chilled collection tubes, chilling the samples in an ice bath, immediate separation of plasma from red blood cells and immediate analysis for ammonia concentration. The ion exchange method was determined to be sufficiently accurate for clinical use and is suggested for any veterinary clinical pathology laboratory.

Journal Article↗
PubMed1979

Separation of oligonucleotides on ion-exchange derivatives of Spheron.

Diphenylamine-formic acid hydrolyzate of Bacillus subtilis DNA (a mixture of pyrimidine oligodeoxyribonucleotides) was chromatographed using an ionic strength gradient on seven ion exchangers: five DEAE-Spherons 300 of various capacities, two types of BD-Spheron 300 and DEAE-Sephadex A-25. The suitability of ion-exchange derivatives of Spheron for the chromatography of oligonucleotides is discussed. The separation of mono- to undeca-deoxyribonucleotides was achieved.

Bacillus subtilis↗
PubMed2004

Purification of hemoglobin by ion exchange chromatography in flow-through mode with PEG as an escort.

Development of hemoglobin-based blood substitutes requires production of highly purified hemoglobin. Process of hemoglobin purification by ion exchange chromatography in flow-through mode was researched and optimized. Three kinds of media including, QMA Spherosil LS (Biosepra, France) and Q Sepharose Big Beads (Amersham Bioscience, Sweden), and an anion exchange membrane column, Mustang Q (PALL, USA) were investigated and compared. Adding polyethylene glycol (PEG) as an escort in ion exchange chromatography improved the purity and recovery, and the recovery in the chromatography was increased from 75 to 95%. The mechanism of PEG effects on chromatography was discussed. The optimal chromatography step, in combination with hypotonic dilution hemolyzing and membrane separation, formed an integrated hemoglobin purification process. The total recovery in the process was 87.6%. The activity of hemoglobin was well preserved: P50 23.2 mmHg, and Hill coefficient 2.31. The product appeared as a single band in SDS-PAGE, and GF-HPLC showed only one peak. The purity of the prepared hemoglobin was more than 99.9%. The optimized process is time saving and suitable for large-scale preparation of hemoglobin to provide materials for further preparation of blood substitutes.

Animals↗