Biomedical subjects

T Scheper

Publications and source records attributed to T Scheper.

54 records · Page 3Linked to original sources

PubMed1993

On-line immunoanalysis for bioprocess control.

Immunoanalytical techniques such as ELISA are often used for the detection of proteins produced in cultivation processes. Owing to the difficulty of automating of the time-consuming traditional ELISA, there is an intense demand for a suitable on-line monitoring method. Combining well-known immunoassays with the FIA technique, we present the heterogeneous and the turbidimetric immuno-FIA methods. The following proteins were investigated with these FIA methods: thermostable pullulanase, IgG, antithrombin III, and recombinant tissue-type plasminogen activator. In the cases of pullulanase and monoclonal mouse IgG, the turbidimetric immuno-FIA was used for on-line analysis of the cultivation process. Results are presented here to demonstrate the effectiveness and application of these immunoanalysis.

Antithrombin III↗

PubMed1992

An integrated process for the production and biotransformation of penicillin.

The extraction of Penicillin G (PG) from the filtered cultivation medium of Penicillium chrysogenum and its conversion into 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PhA) at pH 8 was performed in a 10 l kühni extractor during the production by means of penicillin-G-amidase immobilized in a liquid membrane carrier system (LM). 6-APA was enriched in LM, and the PhA returned to the cultivation medium. After electrocoalescence of LM, the 6-APA was converted into ampicillin with the same enzyme at pH 6, while the liquid membrane phase and enzyme were recycled and reused.

Ampicillin↗

PubMed1992

Immuno- and flow cytometric analytical methods for biotechnological research and process monitoring.

In this article, the applications of immunoanalysis and flow cytometry for research and process monitoring in biotechnology are discussed. Brief reviews of the two analytical methods are followed by descriptions of actual applications in various areas of biotechnology. In the case of immunoanalysis, emphasis is placed on systems for on-line bioprocess monitoring, and examples are given for a thermostable pullulanase, a mouse IgG, and antithrombin III. Although flow cytometry is not currently an on-line analytical technique, its value as an off-line method is illustrated by examples of the measurement of shear stress effects, lipid content, and sterol content.

Animals↗

PubMed1991

Monitoring and control of biotechnological production processes by Bio-FET-FIA-sensors.

Single and multisensor field effect transistors (FET) with a pH-sensitive Si/SiO2/Si3N4/Ta2O5-gate and reference electrode (for single sensor) were developed and used for manufacturing the following biological (Bio)-FETs: for glucose analysis, glucose oxidase-FET (GOD-FET); for urea analysis, urease-FET; and for cephalosporin C analysis, cephalosporinase-FET. The GOD-FETs were integrated into flow injection analysis (FIA) of the Eppendorf variables analyser (EVA) system and used for monitoring the glucose concentration in microbial cultivation and production processes with recombinant Escherichia coli K12 MF, recombinant E. coli JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was used to monitor the urea concentration in a simulated cultivation of Cephalosporium acremonium and urease-FET-FIA and GOD-FET-FIA for the monitoring of urea and glucose concentrations in simulated S. cerevisiae cultivations.

Acremonium↗

PubMed1991

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA.

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product analysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was successfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.

Animals↗

PubMed1991

Metabolic denitrosation of N-nitroso-N-methylaniline: detection of amine-metabolites.

The enzymatic denitrosation of N-nitroso-N-methylaniline (NMA) was investigated by measuring the resulting amine metabolites when NMA was incubated with liver microsomes of PB-pretreated mice. Aniline was found to be the main amine metabolite. Small amounts of the secondary amine, N-methylaniline (MA) and its metabolite, p-methylaminophenol (p-MAP), could also be detected. Incubation of MA resulted in the formation of aniline and p-MAP. The velocity of the metabolism of MA was somewhat faster than that of NMA. On the basis of the measured Vmax values the formation of aniline from MA or from NMA proceeded at nearly identical rates. The dissociation constants as a measure of binding affinity to cytochrome (cyt.) P-450 were determined by measuring the binding spectra. NMA has one Ks of 3.1 mM, whereas MA shows two apparent Ks values, 650 microM and 25 mM, respectively. The results are discussed in relation to the enzymatic mechanism of denitrosation of NMA.

Aniline Compounds↗

PubMed1991

Metabolism of hybridoma cells and antibody secretion at high cell densities in dialysis tubing.

The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter [10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness] inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation. The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily. The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks. The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions. Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities. In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes.

An on-line assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of 1 mg l-1 together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.

Antigen-Antibody Complex↗

PubMed1991

Metabolic denitrosation of N-nitrosamines: mechanism and biological consequences.

NADPH-dependent microsomal metabolism of N-nitrosamines results in both oxidative dealkylation and denitrosation of the molecule. For denitrosation, two enzymatic mechanisms have been proposed: (i) cytochrome P450 (P450)-dependent one-electron reduction of the nitrosamine molecule, resulting in the formation of nitric oxide (NO) and secondary and primary amine, and (ii) liberation of NO via an oxidative mechanism mediated by a P450-dependent one-electron abstraction. In order to clarify the mechanism of denitrosation, the metabolism and kinetics of N-nitrosodibenzylamine (NDBzA) and its corresponding secondary amine dibenzylamine were studied. The main metabolites of NDBzA are benzaldehyde, the primary amine benzylamine and nitrite. An important finding is that benzaldehyde is generated more rapidly from dibenzylamine than from the parent NDBzA. During reductive denitrosation of NDBzA, the oxygen atom in benzaldehyde is derived from air, while benzaldehyde generated via the oxidative mechanism of denitrosation receives its oxygen atom from water due to hydrolysis of the intermediary benzylidenebenzylamine. Microsomal incubation of NDBzA in buffer containing 18O-H2O resulted in no incorporation of 18O from water into benzaldehyde, which could be related to the formation of the corresponding benzaldehyde, which could be related to the formation of the corresponding benzylidenebenzylamine. It is concluded that NDBzA is denitrosated by the proposed reductive mechanism. Current belief is that denitrosation leads to detoxification of the NA molecule; however, toxic effects cannot be excluded if the conversion of NO into NO2- and NO3- involves intermediary formation of the NO2 radical.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotransformation↗

PubMed1987

Enzymatic denitrosation of diphenylnitrosamine: activation or inactivation?

Animals↗

PubMed1987

Metabolic denitrosation of diphenylnitrosamine: a possible bioactivation pathway.

Nitrosodiphenylamine was tested for induction of DNA single strand breaks in rat hepatocytes and Chinese hamster V 79 cells with the alkaline filter elution assay. While in rat hepatocytes DNA damage was observed, negative results were obtained in V 79 cells. In view of the metabolic capacity of hepatocytes and the chemical structure of nitrosodiphenylamine it seems likely that cytochrome P-450-dependent, reductive denitrosation might be necessary for exerting this effect. Therefore the metabolism of nitrosodiphenylamine was investigated in phenobarbital-induced mouse liver microsomes and some of the metabolites were also tested. One metabolite was identified as diphenylamine whereas the others were identified as a ring-hydroxylated derivative of diphenylamine and its corresponding quinoneimine. Diphenylhydroxylamine which was not detected in the microsomes as a metabolite produced a significant amount of DNA single strand breaks in V 79 cells. When diphenylhydroxylamine was incubated with microsomes electron spin resonance spectrum was observed which indicated the formation of the diphenylnitroxide radical. This radical seems to be mediated by auto-oxidation rather than by enzymatic catalysis. Whether diphenylhydroxylamine might be responsible for the observed genetoxic effects of nitrosodiphenylamine assumed to be produced via active oxygen species is discussed.

Animals↗

PubMed1987

On-line measurement of culture fluorescence for process monitoring and control of biotechnological processes.

Explore the source record for details and available documents.

Bacteria↗

PubMed1987

Liquid surfactant membrane emulsions. A new technique for enzyme immobilization.

Liquid membrane reactors are well known for metal extraction. This technology may also be applied to the immobilization of enzymes in enzyme emulsions. The use of liquid membrane reactors for enzymatic bioconversions has several advantages in comparison to solid membrane reactors and conventional immobilization techniques: there is no membrane fouling, enzyme emulsions can be used in cell-free fermentation broths, in complex mixtures the membrane can preselect the desired substrate for enzymatic reaction, and substances that might decrease the enzyme activity can be excluded. The separation effect is not based on differences in molecular weight but on the chemical behavior of the substances to be separated. Thus, it is not necessary to use cofactors with increased molecular weight for enzymatic reactions, since the coenzyme cannot permeate the liquid membrane. The three systems presented here indicate that enzyme systems can be easily immobilized in liquid surfactant membrane emulsions and there is a broad field of application for enzyme emulsions.

Amidohydrolases↗

PubMed1987

Some aspects of cytochrome P450-dependent denitrosation of N-nitrosamines.

The present paper deals with three aspects of cytochrome P450-dependent denitrosation of N-nitrosamines. (1) Nitrate was found in addition to nitrite as a metabolic product of the denitrosation reaction when N-nitrosamines were incubated with a microsomal system. This could also be shown when nitric oxide was added to the microsomes. (2) In order to determine the amount of denitrosation in vivo, the nitroso group of N-nitroso-N-methylaniline was labelled with the 15N isotope and administered to rats; then, the concentrations of 15N-nitrate and 15-N-nitrite in the urine were quantified by measuring the reaction of nitrate and benzene to nitrobenzene. It is estimated from these data that about 33% of the applied dose of 15N-nitroso-N-methylaniline is denitrosated in vivo. (3) Although N-nitrosodiphenylamine (NDPhA) has been classified as a noncarcinogen, recent long-term and short-term studies have cast some doubt. In order to evaluate the mechanism by which NDPhA exerts its possible genetoxic effects, its metabolism was studied in vitro, and NDPhA and its metabolites were tested for induction of DNA single-strand breaks in rat hepatocytes and in Chinese hamster V79 cells. One metabolite was identified as diphenylamine; others were suspected to be the 4-hydroxylated derivative and its corresponding quinoneimine. NDPhA caused DNA damage in rat hepatocytes but not in V79 cells. Diphenylamine also gave negative results in V79 cells, but its putative metabolite, diphenylhydroxylamine, induced a significant increase in DNA single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Biosensors for process monitoring.

A short review about the biosensor research activities for bioprocess monitoring in the F.R.G. after its reunification is given. The principles of biosensor applications are presented. In situ sensors and sensors based on the principles of flow injection analysis are studied. Some applications of a four-channel enzyme thermistor, bio-field effect transistors, and immunoanalysis systems for real process monitoring are presented.

Biosensing Techniques↗

PubMedDate not supplied

Novel applications of fluorescence sensors.

Typically, NAD(P)H-sensitive culture probes have been used to estimate biomass concentrations in suspended-cell cultivations, but these sensors have other uses as well. A number of applications, ranging from biosensors to immobilized-cell metabolic studies, are presented.

Alanine Transaminase↗

PubMedDate not supplied

Application of scanning fluorometry for monitoring of a fermentation process.

Conventional observation of a fermentation process by using NAD(P)H-dependent fluorescence provides a mean for cell monitoring, but its practical application has been very limited due to many factors that affect culture fluorescence. In this work we studied correlation of scanning fluorometry signals with various process variables and with cell metabolic states and showed that scanning fluorometry is more informative than conventional NAD(P)H-dependent fluorescence. A stepwise multiple-regression procedure has been used to select meaningful signals from the whole spectrum, and we found that with three or four components of the excitation-emission plane the process variables can be portrayed with a rather good accuracy. The approach is demonstrated on the examples of wild-type or recombinant Escherichia coli and Saccharomyces cerevisiae fermentations.

Anti-Bacterial Agents↗

PubMedDate not supplied

Two-dimensional fluorescence spectroscopy: a new tool for on-line bioprocess monitoring.

Two-dimensional fluorescence spectroscopy is presented as a new method for bioprocess monitoring. It covers a wide range of excitation and emission wavelengths and is a further development of the fluorescence measurements performed so far, which concentrated mainly on NAD(P)H culture fluorescence. Biogenic fluorophores such as proteins, coenzymes, and vitamins can simultaneously be detected qualitatively and quantitatively inside and outside the cells. This optical method is noninvasive, suitable for in vivo measurements. One whole spectrum (excitation, 250-550 nm; emission, 260-600 nm) with the described parameters is performed within 1 min, which allows an almost continuous monitoring of the bioprocess. The technique is ideal for on-line, in situ measurements via fiber optical systems. Results are presented for cultivations of Claviceps purpurea, Escherichia coli, Saccharomyces cerevisiae, and Sphingomonas yanoikuyae. Cell growth and the metabolism of the cells (changes from aerobic to anaerobic conditions and uncoupling of the oxidative phosphorylation) could be detected.

Biotechnology↗