Biomedical subjects

A Jungbauer

Publications and source records attributed to A Jungbauer.

At least 55 records · Page 3Linked to original sources

PubMed1999

17 beta-estradiol: behavior during waste water analyses.

The distribution between the aqueous and solid phase of 17 beta-estradiol in the ng-range in waste water was investigated by spiking experiments with a radio-labeled hormone. The distribution was measured by liquid scintillation counting. The major part of 17 beta-estradiol remained in the aqueous phase and did not adsorb to the solids. The fraction in the aqueous phase could be enriched by a conventional solid phase extraction with a recovery rate of up to 100%. Losses were negligible and the compound did not adsorb to the surfaces of glass bottles.

Adsorption↗

PubMed1998

Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density.

A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.

Antibodies, Monoclonal↗

PubMed1998

Peptide affinity chromatography of human clotting factor VIII. Screening of the vWF-binding domain.

The region of von Willebrand factor, which is involved in the complex formation with factor VIII, was used to generate a panel of octapeptides. A peptide ladder was generated from the von Willebrand factor region aa40 to aa100 and was synthesized on cellulose membranes by spot technology. Four peptides with affinity for factor VIII were identified by incubation with plasma derived factor VIII and recombinant factor VIII. The peptides denoted as 010 (LCPPGMVRHE), 011 (RCPCFHQGK), 014 (CFHQGKEYA) and 015 (RDRKWNCTDHVC) were further characterized by real-time interaction analysis and small scale affinity chromatography. Biotinylated peptides were used for blotting assays. These experiments showed that the peptides are directed against the light chain of FVIII. We consider these peptides as valuable tools for in situ labeling and also as ligands suitable for affinity chromatography.

Amino Acid Sequence↗

PubMed1998

Bovine whey fractionation based on cation-exchange chromatography.

Bovine whey proteins have potential applications in veterinary medicine, food industry and as supplements for cell culture media. A fractionation scheme for the economically interesting proteins, such as IgG, lactoferrin and lactoperoxidase, based on cation exchangers was the goal of our investigations. A chromatographic process was developed where alpha-lactalbumin passes through the column and separation of the desired proteins is achieved. Four different cation-exchange media (S-HyperD-F, S-Sepharose FF, Fractogel EMD SO3- 650 (S) and Macro-Prep High S Support) were compared in regard to their dynamic binding capacity for IgG and their different elution behaviours when sequential step gradients with NaCl buffers were applied. Peak fractions were analyzed by size-exclusion chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Lactoperoxidase activity was monitored by the oxidation of o-phenylenediamine. In order to explain the different resolution behaviours, isocratic runs with pure standards of whey proteins were performed. The k' values were calculated and plotted against salt concentration. Fractogel EMD had the highest binding capacity for IgG, 3.7 mg/ml gel at a linear flow-rate of 100 cm/h, but the resolution was low compared to that with the other three media. S-Hyper D and S-Sepharose FF showed lower capacities, 3.3 and 3.2 mg/ml gel, respectively, but exhibited better protein resolution. These effects could be partially explained by the k' versus salt concentration plots. The binding capacity of Macro-Prep S was considerably lower compared to that of the other resins investigated because its selectivity for whey proteins was completely different. S-Sepharose FF and S-Hyper D combine relatively high dynamic capacity for IgG and good resolution. Compared to studies with standard proteins, such as 100 mg/ml bovine serum albumin for S-Hyper D, their binding capacities were very low. Even after removal of low-molecular-mass compounds, the capacity could not be improved significantly. The running conditions (low pH) were responsible for the low protein binding capacity, since low-molecular-mass compounds in the feed do not compete with the adsorption of whey protein. The dynamic capacity did not decrease to a large extent within the range of flow-rates (100-600 cm/h) investigated. The dynamic capacity of HyperD and Fractogel was at least five times higher when pure bovine IgG was used for determination. In conclusion, S-Sepharose FF, S-Hyper D-F and Fractogel EMD SO3- 650 (S) are considered as successful candidates for the large-scale purification of bovine whey proteins.

Animals↗

PubMed1998

Regulation of human estrogen receptor by phytoestrogens in yeast and human cells.

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estrogen. They are present in many foods and their higher consumption in certain populations has been correlated with protection against many diseases including coronary heart disease, breast cancer and endometrial and ovarian cancer. In this report, ten phytoestrogens with diverse chemical structures were studied for their binding to the human estrogen receptor and their transcription activation properties in yeast and mammalian cells. Our results showed that some of these compounds bind with relatively high affinity to the estrogen receptor and activate the receptor in the yeast and mammalian cell system. In addition, none of these compounds showed anti-estrogenic activity. We conclude that the yeast system accurately predicts the estrogenic activity of compounds with diverse chemical structures in mammalian cells. In addition, our data with phytoestrogens that do not show transcription activation properties raise the possibility that these compounds may exert their biological effects through pathways different from the classical estrogen signalling mechanism.

Binding, Competitive↗

PubMed1996

Cytokine activity assay by means of proliferation measured in plane convex microtiter wells.

A new assay to measure cell proliferation by turbidimetric evaluation of cultures in specially designed microtiter plates was set up. The IL-2-dependent proliferation of CTLL-2 cell line was used as a model. The novel microtiter plate detection system, the General Cell Screening System (GCSS) was used for the evaluation of the cell proliferation assay. The test system utilizes the population growth rate (mu) of the cells. The tests were performed in specially designed microtiter plates which were read in a scanning spectrophotometer. These results were compared with colorimetric assays, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as substrates. In contrast to conventional methods, the presently described system is not an end-point method, this means that cell growth can be observed over a certain period and is not limited to a single moment during the period of cell growth. Another advantage is the reduction of manual manipulations and the avoidance of toxic or radioactive substances and organic solvents. Therefore, a higher number of samples can be analyzed compared to other methods. The range for detection was 0.015-10 units IL-2 per well, and the accuracy is in the range of < 0.005 standard deviation.

Animals↗

PubMed1996

Structural and functional analysis of N-terminal point mutants of the human estrogen receptor.

Twenty N-terminal point mutations of the human estrogen receptor (hER) were constructed as ubiquitin fusion products and expressed under the control of the copper regulated promoter CUP1 in Saccharomyces cerevisiae. The objective of these studies was to overexpress hER in yeast and also to evaluate the functional properties of the N-terminal variants of hER. Fusion of the C-terminus of ubiquitin to the N-terminus of other proteins has been shown to increase the level of protein expression in yeast. Ubiquitin C-terminal hydrolases (UCHs) in yeast efficiently and precisely cleave at the junction with ubiquitin and render free hER with desired amino termini. The variant hER proteins, that were generated by mutating the N-terminus of hER, showed enormous differences in receptor protein levels and transactivation potential. All variant hER proteins were synthesized as 66 kDa species as identified by Western blotting with the exception of the proline-containing variant (Pro-ER). The UB-Pro-ER variant was cleaved inefficiently by UCHs in yeast. The UB-Pro-hER [correction of UB-Pro-hEr] variant also exhibited a different DNA band-shift profile compared to those of the other receptor variants and the wild-type. Val-, Thr-, and Lys-ER did not express, as measured by enzyme-immunoassay and Western blotting; nor did they transactivate a beta-galactosidase reporter gene in yeast. However, the Glu-ER was 50% more active in transactivation as compared to the wild-type. The results of the receptor content, DNA binding properties and transactivation analysis in yeast demonstrate that the N-terminal residue plays an important role in the structure and function of hER.

Amino Acid Sequence↗

PubMed1995

Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.

Chromatography↗

PubMed1995

DNA clearance in chromatography of proteins, exemplified by affinity chromatography.

Complications of DNA clearance in protein chromatography using the conventional methodology of spiking experiments are reported. Protein A affinity chromatography demonstrated this complications in a small scale experiment. A concentrated hybridoma culture supernatant was spiked with DNA extracted from hybridoma cells fed with [3H]thymidine. Protein A affinity chromatography was subsequently carried out. The column effluent was collected in fractions, and each fraction was analyzed for radioactivity and IgG levels. A substantial amount of DNA was eluted before the main IgG peak. Frequently a small peak is observed in front of the main peak in protein chromatography. This phenomenon can be explained by either displacement effects, or incomplete washing, or hysteresis during the adsorption and desorption conditions. Fractionation at the beginning of elution is critical to the maintenance of a high standard protein purity.

Chromatography, Affinity↗

PubMed1994

Manufacture of recombinant proteins with safe and validated chromatographic sorbents.

Purification of recombinant proteins to achieve homogeneity, purity, consistency and potency as required for therapeutic proteins and in vivo diagnostics is performed under stringent and validated conditions. As liquid chromatography is one of the major technologies used for this purpose, it has to be carried out according to special regulatory guidelines. One of the reported aspects is the long-term consistency of a chromatographic process and validation of its operation; other aspects described are more sorbent oriented. In-place cleaning and sterilization are also very important aspects, the efficiency of which is dependent on the chosen working conditions and the chemical nature of the sorbents. Drastic cleaning may deteriorate the chromatographic matrices, releasing chemicals that may contaminate the biologicals of interest, which modifying the behaviour of the chromatographic columns. Moreover, leachable compounds, when present, could have adverse effects in case of high toxicity. Determination of leaching levels and toxicity tests are part of the validation steps to turn chromatographic separations into consistent, effective and safe production processes for biologicals.

Animals↗

PubMed1994

Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes.

Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N-terminal acetylation or glycosylation should be ruled out. When purified by small-scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material. Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2-SOD, as reported in the literature. Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190-240 nm range, but marked differences in the 250-350 nm region. Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.

Circular Dichroism↗

PubMed1994

A broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1.

We have established a hybridoma clone, designated 2F5, secreting a neutralizing human monoclonal antibody (MAb) specific for gp41 of human immunodeficiency virus type 1 (HIV-1). The epitope of MAb 2F5 was mapped to amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala on the ectodomain of gp41. In this study different in vitro test systems were used to characterize the neutralizing properties of MAb 2F5. In syncytium inhibition assays, fusion inhibition experiments, and neutralization assays on different HIV-susceptible cells (H9, U937, and peripheral blood mononuclear cells) MAb 2F5 showed broad-spectrum neutralizing capacity against HIV-1 laboratory isolates IIIB, MN, RF, and SF2. In addition, primary isolates from AIDS patients were also neutralized.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization.

Electrofusion and EBV transformation were studied by immortalizing human PBLs from blood of HIV-1-positive volunteers. A panel of 33 cell lines producing human monoclonal antibodies (Hu-MAbs) against HIV-1 was established by cell fusion or EBV transformation. For the first fusion experiments the source of B lymphocytes was peripheral blood of HIV-1-infected donors in CDC stages II or III with CD4 cell counts higher than 500/mm3. Later on, from these patients only, those with high anti-HIV titers were chosen as blood donors. By that means the yield of stable specific hybridomas was increased twofold. In our experiments electrofusion turned out to be a more efficient immortalization method than EBV transformation, due to a high and constant immortalization rate. The hybridomas were stable after intensive subcloning and could be cultivated over a period of 8 months without loss in monoclonal antibody production. Immunoglobulin class, subtype, reactivity against HIV-1 proteins, Western blot patterns, immunofluorescence, and epitopes were characterized. The subtype of all antibodies was IgG1 or IgG3. The light chain was predominantly kappa. All antibodies showed reactivity against HIV-1 envelope or core protein. All hybridomas were stable and suited for mass production. Several Hu-MAbs are becoming an important tool in the field of diagnosis, research, and immunotherapy.

Antibodies, Monoclonal↗

PubMed1994

Stable, continuous large-scale production of human monoclonal HIV-1 antibody using a computer-controlled pilot plant.

A completely automated pilot plant used for fermentation has been employed with direct digital control (DDC) technology for monitoring and regulating growth of human cells. A human hybridoma cell line (3D6) producing anti-human immunodeficiency virus (HIV)-1 antibodies was used as a model for large-scale production (300-liter airlift fermentor) in continuous culture. Parameters controlled were pH, dissolved oxygen, temperature and the flow rate of four gases used in the process. A control strategy was implemented to achieve constant fluid velocity and mixing by maintaining the rate of gas flow at a constant level. Another advantage of this approach was that the total gas flow required for optimal fluid circulation was reduced from 1 volume gas/volume fermenter/hour (vvh) to 0.3 vvh. Use of a low flow rate eliminated the serious problems of foaming, which contributed significantly to cell destruction, shorter filter-life and other considerations. Dilution rate was optimized at laboratory scale for maximum productivity, which results in relatively low viability. At a dilution rate of 0.0076 h-1, a total cell density of 6-7 x 10(5) cells/ml with a viability of approximately 75% was maintained during long-term continuous cultivation. These growth conditions resulted in a product titer stabilized in the range of 35 micrograms IgG/ml. Batchwise purification was achieved with a recovery of more than 50% and a final purification of active monoclonal antibody representing about 99% product. Results from isoelectric focusing and Western blotting demonstrated batch-to-batch consistency of the purified human monoclonal antibody to HIV-1 during the continuous growth process.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1993

Determination of immune complexes by high-performance gel chromatography (positive cooperativity of antibody-antigen reaction).

Positive cooperativity of antibody-antigen complex formation using human Cu/Zn superoxide dismutase and a murine monoclonal antibody as model is demonstrated by high-performance gel chromatography using TSK G3000sw and TSK G4000sw columns. Three different antibody-antigen complexes named Type I, II and III composed from two antigen molecules+two antibody molecules, two antigen molecules+one antibody molecule and one antigen molecule+one antibody molecule could be separated. The degree and ratio of complexes present in solution depends mainly on the ratios of antibody and antigen.

Animals↗

PubMed1992

Scale-up of recombinant protein purification by hydrophobic interaction chromatography.

The scale-up of hydrophobic interaction chromatography is described. Human recombinant superoxide dismutase was used as a model. The scale-up was performed by keeping the height to diameter (H/D) ratio of the column constant. The success of scale-up was evaluated by reversed-phase high-performance liquid chromatography of the eluted material. The wrong H/D ratio causes decreased resolution.

Chromatography, High Pressure Liquid↗

PubMed1990

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I.

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable plate-like crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

Antibodies, Monoclonal↗

PubMed1990

Capillary zone electrophoresis for monitoring r-DNA protein purification in multi-compartment electrolysers with immobiline membranes.

Isoforms of human monoclonal antibodies against the gp-41 of AIDS virus and of human recombinant superoxide dismutase have been purified to homogeneity by isoelectric focusing (IEF) in a multi-compartment electrolyser with isoelectric, immobiline membranes. This system allows the processing of large sample volumes and gram-scale protein loads and can resolve isoforms as close as 0.001 in pI difference. The purification progress was usually monitored by analytical IEF in immobilized pH gradients (IPG). Capillary zone electrophoresis (CZE) was applied to the monitoring of the content of each chamber of the electrolyser. CZE was found to be superior in terms of speed of analysis and quantification (but only by UV reading at 200-210 nm, i.e., in the region of the peptide bond) but, notwithstanding the millions of theoretical plates reported, was no match for the resolving power of IPGs, at least for protein analysis. When compared also with chromatofocusing, the resolving power decreases in the order IPG greater than CZE much greater than chromatofocusing.

Antibodies, Monoclonal↗