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R Freitag

Publications and source records attributed to R Freitag.

At least 37 records · Page 2Linked to original sources
PubMed1998

Development of a downstream process for the isolation and separation of monoclonal immunoglobulin A monomers, dimers and polymers from cell culture supernatant.

The isolation and separation of the molecular variants of monoclonal IgA from cell culture supernatants is possible using several filtration and ion-exchange chromatography steps, followed by size-exclusion chromatography for the actual separation of the molecular variants. The latter step is especially time consuming and laborious. This report presents possible improvements of the procedure. Use of the displacement rather than the elution mode may render the ion-exchange step more productive (higher product concentrations and space-time yield). For the final separation of the molecular variants, hydroxyapatite (HA) elution chromatography can serve as an alternative to size-exclusion chromatography. By using an optimized, complex phosphate gradient, the IgA dimers can be separated quantitatively from the monomers and higher oligomers. It may in individual cases be necessary to use a size-exclusion polishing step to reach the required final degree of purity, however, the amount of material to be processed is reduced to such an extend by the HA-step, that the overall process is still more productive. Buffer pH and flow-rate as well as the stationary phase material used were additional factors considered during the optimization of the HA elution chromatography. HA-displacement chromatography resulted only in a concentration of the overall IgA fraction, but not in a separation of the molecular forms.

Animals↗
PubMed1998

Psychosocial aspects of child abuse for primary care pediatricians.

Childhood is an image that advertisers use to conjure up feelings of care-free innocence. Society frequently calls for protecting our children and creating strong, healthy families. Yet thousands of children experience violence on a regular basis, and their lives are irretrievably altered. For these children, the sites of violence and not war-torn villages or crime-ridden streets but their own homes. This article provides primary care physicians with basic information about the psychosocial aspects of recognition and prevention of child abuse.

Child↗
PubMed1998

Displacement chromatography using the UNO continuous bed column as a stationary phase

Displacement separations of biopolymers are even more restricted in regard to the upper applicable limit of the flow rate than elution chromatographic ones, when conventional columns packed with porous particles are used. Bio-Rad has recently introduced a new column type, the UNO column, which contrary to conventional HPLC columns is not packed with particles but consists of a continuous porous polymer rod. The plate height of this continuous bed column was found to be nearly independent of the flow rate within the investigated range (0.01-4.5 mL/min, 1.56-701.6 cm/h). The strong ion exchanger columns UNO Q1 (7 x 35 mm, 1.3 mL) and UNO Q6 (12 x 53 mm, 6 mL) were investigated as stationary phases for protein displacement chromatography at elevated flow rates. With the UNO column, displacement separations of alpha-lactalbumin and beta-lactoglobulin (displacer: poly(acrylic acid), Mw 5100) were still possible at flow rates that were 1 order of magnitude higher than those applicable to conventional columns of similar dimensions. In fact the flow rate was limited by the necessity to collect the protein-containing fraction fast enough for an adequate monitoring of the separation rather than by a loss in resolution. While the UNO Q1 column did not allow for the full development of the displacement train, 50 mg of protein could be separated using the UNO Q 6 column. Recoveries were well over 95% in this case. More than 50% of the alpha-lactalbumin was collected in pure and concentrated form (concentration by a factor of 2). The steric mass action model was used to optimize the displacer concentration.

Journal Article↗
PubMed1997

Comparison of anion-exchange and hydroxyapatite displacement chromatography for the isolation of whey proteins.

In displacement chromatography, several substances may be isolated and concomitantly concentrated, which makes this separation procedure attractive for the processing of diluted product streams containing a number of high value substances. Here, the suitability of anion-exchange and hydroxyapatite displacement chromatography for the processing of technical dairy whey is investigated. The pH and flow-rate of the carrier, the displacer chemistry and, in case of the apatite, the particle diameter of the stationary phase are considered. As a consequence of the pH sensitivity of the beta-lactoglobulin, one major whey component, apatite displacement chromatography is less then successful in whey separation. At a denaturing carrier pH (> 8.5) the beta-lactoglobulin zone is broad and stretches over the entire displacement train. At a lower carrier pH, previously successful polyanionic displacers do not bring about separation, while low-molecular-mass ones do, but they tend to overrun and thus contaminate the protein zones. In the case of anion-exchange displacement chromatography, polyanions, especially polyacrylic acid (PAA, M(r) 6000), constitute suitable displacers. Here too, a carrier pH of 8.0 is most suited to the separation of the whey proteins. The low-molecular mass-displacer iminodiacetic acid (IDA, M(r) 133.4), on the other hand, displaces only alpha-lactalbumin. The beta-lactoglobulin remains on the column. PAA is used as the displacer to process a dairy whey sample.

Animals↗
PubMed1997

Analysis of endotoxins by capillary electrophoresis.

Endotoxins are part of the outer membrane of gram-negative bacteria such as E. coli. Upon entering the blood stream, they cause a violent, sometimes life-threatening, response of the immune system. Endotoxins are lipopolysaccharides (LPS), lacking optically active groups, and their detection in the underivatized state can be difficult. In this paper the potential of capillary electrophoresis (CE) for LPS analysis is investigated. By using a standard phosphate buffer method, concentrations down to 100 micrograms/mL can be detected within 6 min. The detection limit can be lowered by one order of magnitude by using a sodium dodecyl sulfate (SDS)/borate buffer, pH 9.2. In this buffer, the SDS serves to homogenize the size of the LPS aggregates, while the borate forms complexes with the diol groups of the molecule, thereby enhancing its optical activity. The formation of LPS-affinity complexes with the UV-active polymyxin B or labeling of the LPS with a fluorophore (fluorescein isothiocyanate) was unsuccessful. Best results, in terms of detection limit and speed, were obtained with an indirect UV-detection CE method. By using a strongly UV-active electrophoresis buffer, endotoxins could be detected as "negative" peaks. In this case, a detection limit of 3 micrograms/mL (35 pM) was determined. Proteins and other UV-active substances did not disturb the assay, since they generated no detectable signals. The indirect UV detection was used to quantify the residual LPS content of a DNA preparation from E. coli.

Carbohydrate Sequence↗
PubMed1996

Analysis of amatoxins alpha-amanitin and beta-amanitin in toadstool extracts and body fluids by capillary zone electrophoresis with photodiode array detection.

Over 90% of the lethal cases of mushroom toxin poisoning in man are caused by a species of amanita. The amatoxins (especially alpha- and beta-amanitin) found in amanita deserve special attention, because of their high pharmacological potency, their high natural concentration and their high chemical and thermal stability. Measures can be taken to improve the survival rates (aggressive gastroenteric decontamination, liver protection therapy) if the poisoning is diagnosed correctly and as early as possible. The standard assay for alpha-amanitin is a radioimmunoassay (RIA). Among other reagents, this assay uses 125I-labelled alpha-amaintin, which has a low shelf life. The assay is therefore not available at all hospitals and all year round. In this paper, a first attempt to employ capillary zone electrophoresis (CZE) to quantify amatoxins alpha- and beta-amanitin in urine samples of afflicted patients and in toadstool extracts is described. Diode array detection is used for identification of the resolved substances in the electropherogram. An analysis requires 20 min. The detection limit is 1 microgram/ml, i.e., 5 pg absolute. Relative standard deviations are between 1 and 2% for the calibration standards (peak height and area) and ca. 7.5% for the real samples. Advantages of the CZE over the RIA include lower cost, the possibility of quantifying several toxins in one analysis, less consumption of potentially harmful reagents (no radio-labelled substances, no addition of alpha-amanitin as reagent) and, most importantly, all-year-round availability of the assay. The detection limit is still somewhat high and does not cover the entire clinically relevant range. Attempts to lower the detection limit by the necessary order of magnitude are currently under way in our laboratory. These include application of laser-induced fluorescence detection, liquid chromatography-CZE and CZE-mass spectrometry techniques.

Amanita↗
PubMed1996

Preparative membrane adsorber chromatography for the isolation of cow milk components.

Preparative membrane adsorber (MA) chromatography was used to process milk fractions such as the whey and the permeate commonly obtained during lactose production in modern dairies. In MA systems the fluid-dynamic and mass-transfer properties are superior to conventional HPLC or fast protein liquid chromatography (FPLC) columns. Since the flow resistance caused by the MA stacks is quite low, high throughputs can be realized without loss in resolution. Feed sizes were varied from the laboratory scale (several ml) up to batches of 10 1 during the investigations. MAs based on modified cellulose filtration membranes (average single layer thickness 200 microns, average pore size 5.0 microns) were used for the small-scale experiments. The MAs are functionalized by covalent linkage of strong and weak ion exchanger groups to their surface. Three commercially available types were used [strong ion exchanger: MA Q15 (3 layers of 5 cm2) and MA Q100 (5 layers of 20 cm2); weak ion exchanger: MA D15 (3 layers of 5 cm2); all Sartorious, Germany]. For the large-scale work a dead-end filtration unit containing up to 1300 cm2 of MA-area was used. Here MAs based on a synthetic co-polymer, that were prepared from cut-out sheets, were inserted. Chromatographic conditions were transferable from the cellulose- to the polymer-based MA carrying the same functional groups. The influence of the flow-rate and the pH of the mobile phase on the separation was investigated. The flow-rate could be raised to the limit of the respective chromatographic systems and/or MA modules without loss in resolution. The use of the strong anion exchanger MA together with a mobile phase pH of 6.0 and a fine-tuned gradient allowed the separation of BSA, alpha-lactalbumin and the genetic variants of beta-lactoglobulin, even though no baseline separation was possible in the latter case. The use of coupled modules rather than a single one is shown to improve the separation considerably.

Absorption↗
PubMed1996

Chromatography in the downstream processing of biotechnological products.

Chromatography techniques are essential for the isolation and purification of most of the high value products of modern biotechnology. The economically sensible and technically satisfactory downstream processing of a therapeutic protein, usually involves a number of chromatographic steps. Its development and optimization require considerable knowledge of the various physico-chemical and engineering aspects of biochemical chromatography. This review addresses the various modes of chromatography and the design of chromatographic separation processes from a biotechnologist's point of view. Strategies for optimizing the structure of the downstream process are outlined and scaling up consideration are discussed. The importance of the different chromatographic methods in research and development is estimated in an analysis of protein purification schemes recently published in the literature. Finally, examples of the application of chromatographic procedures for process scale product purification in the biotechnological industry are given.

Biotechnology↗
PubMed1996

Evaluation of affinity filters for protein isolation.

Affinity filters were investigated for their potential in the recovery of proteins from complex samples. The experiments covered membranes carrying high and low molecular weight affinity ligands as well as group and substance specific ones. For the ready-to-use affinity filters the specific protein binding capacity was determined and compared to that of the respective Sepharose affinity gels (Pharmacia). In the case of the pre-activated membranes the influence of the coupling chemistry on the affinity mediator concentration and the protein binding capacity were considered in the study. In the case of low molecular weight ligands (e.g. Cibacron Blue, Heparin) either type of membrane yielded stationary phases of a ligand concentration, binding capacity, resolution, and long term stability similar to that of the corresponding Pharmacia material. However, the membranes could be used at a higher flow rate than the columns, since they are less mass transfer limited and cause significantly less back pressure. The immobilization of high molecular weight ligands such as antibodies (immuno filtration) on the other hand, resulted in low ligand concentrations and worse antigen binding capacities whenever conventional immobilization procedures, e.g. epoxy group-based reactions, were used. In contradistinction, good results were obtained with tosyl- and tresyl activated membranes. Such membranes were successfully employed for the immobilization of monoclonal antibodies (mAb) and Concanavalin A. Concanavalin A and an anti gp 220/350 mAb were subsequently used to produce affinity filters for the isolation of a recombinant gp 220/350 Epstein-Barr virus surface antigen from culture supernatants of a Chinese hamster ovary cell line grown in protein-free medium.

Animals↗
PubMed1995

Studies of complexes between proteases, substrates and the protease inhibitor alpha 2-macroglobulin using capillary electrophoresis with laser-induced fluorescence detection.

Capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection is shown to constitute a unique technique for the investigation of the interaction between proteases, protease inhibitors and substrates. Under optimized analysis conditions, the formation of a complex between FITC-labelled proteases such as trypsin, plasmin, alpha-chymotrypsin and the (unlabelled) protease inhibitor alpha 2-macroglobulin was studied. This is not possible with UV detection, since under such conditions the complex cannot be distinguished from the unreacted protease inhibitor. Low ratios of FITC bonded to the proteases further complex formation, while high ratios often prevent the reaction. Complex formation shows a strong dependence on the incubation conditions (pH, salt concentration, temperature, incubation time). Once formed, however, the complexes are stable under CZE conditions (e.g., a pH of the electrophoresis buffer of 10.5) for at least 30 min. Treatment with sodium dodecyl sulfate (5 min at 90 degrees C or 30 min at 75 degrees C) does not destroy the complexes, whereas treatment with mercaptoethanol (reduction of disulfide bonds) eliminates the peak from the electropherogram. Both findings argue for the formation of a covalent bond between the protease and the inhibitor during complex formation. Since the reaction of the proteases with alpha 2-macroglobulin does not involve the binding site of the former, a residual proteolytic activity is still observed in the ensuing complex. The extent of the inhibition of the remaining trypsin activity in a trypsin--alpha 2-macroglobulin complex was established to depend on the molecular mass of the second trypsin inhibitor.

Electrophoresis, Capillary↗
PubMed1995

Quantification of crystal growth of calcium oxalate in gel and its modification by urinary constituents in a new flow model of crystallization.

PURPOSE: To use a new flow model of crystallization to quantify the growth of calcium oxalate monohydrate (COM) in a gel matrix under simulated physiological conditions. MATERIALS AND METHODS: Artificial urine supersaturated in COM was conducted through channels containing a gel with COM seed crystals (flow rate: 0.5 ml. per minute; reaction time: 4 hours; 37 +/- 1C). A relative crystal growth parameter, Vcr, was quantified by discontinuous measurement of scattered light intensity by microphotometry. The effects on Vcr of varying a series of urinary parameters were investigated within the system. RESULTS: The following order of effectiveness in changing COM growth could be derived: dilution > oxalate > calcium > magnesium approximately citrate. CONCLUSIONS: Drinking therapy and lowering of urinary oxalate excretion can be concluded to be the most promising means of preventing CaOx urolithiasis. The new crystallization model demonstrated here should be suitable to new applications in the investigation of urinary stone formation.

Calcium Oxalate↗
PubMed1994

Effect of bed compression on high-performance liquid chromatography columns with gigaporous polymeric packings.

The behavior of chromatographic columns packed with gigaporous, highly cross-linked styrenic particles was investigated for use in protein separation by reversed-phase chromatography at high flow velocities. Stainless-steel columns which were 3.5 or 7.5 cm long and had an inner diameter of 0.46 mm were slurry packed with 8 or 20 mm diameter spherical particles of 4000 A mean pore size by using methanol as the packing fluid. It was found that the conditions employed during the packing process have a dramatic effect on the properties of such columns and that this can be attributed in part to the deformability of the particles. An increase in the packing pressure to approximately 6000 p.s.i. (41 MPa) resulted in a higher mass-transfer efficiency for the column with a concomitant decrease in permeability. This is ascribed to a decrease in the interstitial porosity with increasing packing pressure since the experimentally measured plate heights for these columns were found to agree quantitatively with theoretical predictions that relate changes in the interstitial porosity to intraparticle mass transfer. However, the theoretically derived relationship between porosity, permeability, and efficiency does not hold for columns packed at pressures higher than 6000 p.s.i., in which case the total column porosity was found to be high while the permeability and column efficiency were low. This behavior is explained by the formation of a low-porosity layer of highly compressed particles at the downstream end of the column during high pressure packing so that the assumption of axially uniform column properties used in the theoretical approach leads to very large errors.

Chromatography, High Pressure Liquid↗
PubMed1994

Fluorescein isothiocyanate-labeled protein G as an affinity ligand in affinity/immunocapillary electrophoresis with fluorescence detection.

Antibodies from human sera (h-IgG) were tagged with a fluorescent dye (fluorescein isothiocyanate, FITC) through the affinity reaction of FITC-labeled protein G with the Fc fragment of the antibodies. The complexes were quantified by capillary zone electrophoresis (CZE) within 1 min, i.e., fast enough to prevent their dissociation during the measurement. Conditions for the affinity reaction and the CZE analysis could thus be optimized independently. When an FITC-labeled protein G concentration of 10(-6) mol/L was used, h-IgG concentrations between 10(-6) and 10(-9) mol/L were reproducibly quantified (STD < 2%), using an LIF detector. A correlation coefficient, r2, of 0.9988 was established between the peak height and the IgG concentration. Alternatively, h-IgG containing serum samples and the FITC-labeled protein G were simply injected into the CE capillary in consecutive zones, followed by the application of the electrical field. Within 2 min, the affinity complexes were resolved and the IgG content of the serum quantified (r2 = 0.9986). The injection sequence was of no consequence. The measurements agreed well with those found in a single radial immunodiffusion (SRID) assay. In addition FITC-labeled protein G-tagged anti-h-IgG1 antibodies were used to detect the specific antigen of the involved antibody, namely, h-IgG1, in human sera.

Electrophoresis↗
PubMed1994

Control of the cultivation process of antithrombin III and its characterization by capillary electrophoresis.

The production by baby hamster kidney cells of recombinant antithrombin III (r-AT III), the main inhibitor of thrombin, factor Xa and other proteases of the clotting cascade, was monitored by capillary isotachophoresis using mixtures of continuous spacers. The results were compared with those obtained by capillary zone electrophoresis (CZE). The downstream process, which incorporated anion-exchange and heparin affinity chromatography, was monitored by CZE under acidic conditions and voltage ramping. The purified product was characterized by its isoelectric point and molecular mass. Isoelectric points of the three major and three minor isoforms of AT III were evaluated by capillary isoelectric focusing using a pH range of 4-6 and various mobilization procedures. The molecular mass of AT III was investigated by capillary gel electrophoresis (CGE), applying removable dextran gels. Both parameters could be determined within 30 min using only one coated capillary. The results showed an excellent correspondence with those achieved with conventional slab gels. The affinity complex between AT III and thrombin could also be detected by CGE and the heparin dependence of the affinity reaction could be investigated.

Animals↗
PubMed1994

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Amino Acid Sequence↗
PubMed1994

Comparison of membrane adsorber (MA) based purification schemes for the down-stream processing of recombinant h-AT III.

Several multistage chromatographic separation schemes based on Membrane Adsorbers as stationary phases were designed for the isolation of recombinant human Antithrombin III from supernatants of baby hamster kidney cell cultures. The Antithrombin III concentration of the culture supernatants varied between 6 micrograms/ml and 14 micrograms/ml. The culture media contained 10% foetal calf serum. The concomitant overall protein concentration ranged from 5.4 mg/ml to 6.2 mg/ml, with bovine serum albumin constituting approximately 60%. Strong cation and anion exchanger, Heparin-, and Cibacron Blue Membrane Adsorber were used. While Heparin-Membrane Adsorber were found to isolate and concentrate the Antithrombin III efficiently, the removal of the major bovine serum proteins (albumin, transferrin, immunoglobulins) required a multistage process. By using a sequence of ultrafiltration, diafiltration, Cibacron Blue, anion exchanger, and Heparin-Membrane Adsorber, an electrophoretically pure Antithrombin III could be obtained. Subsequent high sensitivity gel immunoelectrophoresis proved the isolated protein free of bovine IgG and bovine transferrin, while approximately 2% serum albumin could still be discerned. A reduction of the serum content (3%) allowed the isolation of high purity Antithrombin III (> 99.9%), however, the product's specific activity was halved. The Down-Stream-process was designed and optimised for a mobile phase flow rate of 2 ml/min (0.12 l/h). With the exception of the final Heparin affinity step, however, flow rates of up to 4.8 l/h could be used without adverse effect on the final purity, with a concomitant increase of the throughput. Batches of up to 10 1 cell culture supernatant were processed in an automated procedure.

Adsorption↗
PubMed1994

High-performance capillary electrophoresis of human serum and plasma proteins.

In recent years, capillary electrophoresis has proved extremely useful for separation of biomolecules such as nucleotides, amino acids, peptides, and proteins. The advanced instrumental design of commercial CE equipment has offered several advantages to conventional electrophoresis. Precise sample application, temperature regulation, and stable, reproducible instrument control with respect to voltage, current, pressure, and run-to-run reproducibility is routinely achieved in contemporary commercially available equipment. Based on these instrumental features, CE is already used as a routine method for the analysis of small molecules. In contrast, serum and plasma proteins represent a difficult challenge for capillary electrophoresis. Routine analysis of serum and plasma proteins is reduced to the application of capillary zone electrophoresis, which proved to be a reliable method for rapid screening of whole serum, purity control of serum proteins and fractions, as well as on-line quantification of the components. With regard to the simplicity of the method and the low cost of operation, CZE proved to be an attractive alternative to comparable conventional methods. Other separation techniques, such as CITP, CIEF, and CGE, provide higher resolution of the sample proteins than that provided by CZE. Furthermore, information about physicochemical properties of the analyte, such as isoelectric point or molecular weight, can be gained by CIEF or CGE. Even though this information is essential for protein analysis and characterization, these methods are not commonly used at present because reliable methods for controlling the EOF are not commercially available. Coated capillaries are expensive, and it is still difficult to purchase a set of coated capillaries with identical properties. As long as the reproducibility from capillary to capillary cannot be guaranteed, the high resolving methods will not be applied in routine analysis. The development of reliable regulation of the EOF, either by an appropriate column material or coating or an external field control, will certainly lead to a breakthrough for the capillary electrophoresis of human serum and plasma proteins.

Blood Proteins↗