Biomedical subjects

W D Deckwer

Publications and source records attributed to W D Deckwer.

44 records · Page 3Linked to original sources

PubMed1990

Correlation of migration behavior in free-flow zone electrophoresis and electrophoretic titration curve.

The correlation of electrophoretic migration behavior in free-flow zone electrophoresis (FFZE) and electrophoretic titration curve (ETC) has been explored. It is shown that the ETC of a protein or a mixture of proteins can be used to predict the fraction numbers at which those proteins elute in a preparative scale FFZE experiment. The ETC is a quick and effective way to choose optimal buffer conditions in FFZE. FFZE is employed to determine the isoelectric points (pI) of proteins whose pIs lie beyond the range of IEF 3-9 gels. It is found that separations in FFZE are governed by the net surface charge of the proteins.

Buffers↗

PubMed1990

Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).

Alcohol Oxidoreductases↗

PubMed1990

Ambruticin S production in air-lift and stirred-tank bioreactors.

Using the method of equi-inocular synchronized comparative fermentation (EISCF) the cultivation of Sorangium cellulosum So ce 10 and production of the polyketide antibiotic ambruticin S was compared in stirred-tank and air-lift reactors of different geometry. This method requires that inocula originate from the same pre-culture and cultivation parameters are synchronized to similar values. Similar ambruticin yields were obtained from both reactor systems provided that the concentration of dissolved oxygen (DO) was maintained above a certain value (ca. 40%). For cultivation of S. cellulosum it is the DO level rather than the oxygen transfer rate which presents the proper criterion for scale-up and comparative reactor studies.

Alanine↗

PubMed1990

Ambruticin S production in amino acid rich media.

The antimycoticum ambruticin was produced by cultivating the myxobacterium Sorangium cellulosum in shake flasks and stirred tank reactors with 10- and 30-l working volumes. During growth and product formation amino acid concentrations in the complex media were measured by HPLC. In the logarithmic growth phase free aspartate and glutamate were assimilated without product formation. After consumption of aspartate and glutamate, growth was limited and ambruticin production set in. The effects of biological parameters such as age, amount of inoculum, pretreatment of cells before inoculation and time of precultivation were also investigated.

Amino Acids↗

PubMedDate not supplied

A kinetic model for substrate and energy consumption of microbial growth under substrate-sufficient conditions.

The growth of heterotrophic microorganisms can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It is generally observed that the consumption rates of substrate and energy (ATP) are higher under substrate-sufficient conditions than under conditions of substrate limitation. The excess substrate and ATP consumption is often influenced by the residual concentration of substrate in a relatively wide range. To account for these effects, a kinetic model is proposed to describe substrate and ATP consumption rates of microbial growth under substrate-sufficient conditions. According to the model, the specific substrate consumption rate of a substrate-sufficient culture can be expressed as the sum of the substrate rate under substrate-limited conditions at the corresponding specific growth rate and an additional consumption rate due to excess substrate. The same kinetic form also applies to the specific ATP consumption rate and to the specific oxygen consumption rate of an aerobic culture, respectively. The linear equations for substrate and ATP consumption rates of Pirt and of Stouthammer and Bettenhousen can be used for substrate-limited growth. The excess of substrate and ATP consumption rates at carbon surplus can be described in a form similar to that of Michaelis-Menten kinetics. The proposed kinetic model has been verified with experimental data from three continuous cultures representing both anaerobic and aerobic microbial growth on substrates with low and high degrees of reductance. Using this model, the parameters maximum growth yield and maintenance requirement (both in terms of substrate and ATP) of a culture under different growth limitations can be better defined and quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMedDate not supplied

Stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line in batch and continuous cultures.

Batch and continuous cultures were carried out to study the stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line, with particular attention to the metabolism at low levels of glucose and glutamine. The apparent yields of cells on glucose and glutamine, lactate on glucose, and ammonium on glutamine were all found to change significantly at low residual concentrations of glucose (< 5 mmol/L) and glutamine (< 1 mmol/L). The uptake rates of glucose and glutamine were markedly reduced at low concentrations, leading to a more effective utilization of these nutrients for energy metabolism and biosynthesis and reduced formation rates of lactate and ammonium. However, the consumption of other amino acids, especially the essential amino acids leucine, isoleucine, and valine and the nonessential amino acids serine and glutamate, was strongly enhanced at low glutamine concentration. Quantitatively, it was shown that the cellular yields and rates associated with glucose metabolism were primarily determined by the residual glucose concentration, while those associated with glutamine metabolism depended mainly on the residual glutamine. Both experimental results and analysis of the kinetic data with models showed that the glucose metabolism of BHK cells is not affected by glutamine except for a slight influence under glucose limitation and glutaminolysis not by glucose, at least not significantly under the experimental conditions. Compared to hybridoma and other cultured animal cells, the recombinant BHK cell line showed remarkable differences in terms of nutrient sensitivity, stoichiometry, and amino acid metabolism at low levels of nutrients. These cell-line-specific stoichiometry and nutrient needs should be considered when designing an optimal medium and/or feeding strategy for achieving high cell density and high productivity of BHK cells. In this work, a cell density of 1.1 x 10(7) cells/mL was achieved in a conventional continuous culture by using a proper feed medium.

Amino Acids↗

PubMedDate not supplied

Variation of stoichiometric ratios and their correlation for monitoring and control of animal cell cultures.

The stoichiometry of animal cell cultures is examined with respect to its variation and suitability for process monitoring and control. In addition to the two often used stoichiometric ratios, i.e., lactate yield from glucose (Lac/Glc) and ammonium yield from glutamine (NH4+/Gln), five other less well characterzied ones, i.e., ammonium yield from the total consumption of amino acids (NH4+/TAA), consumption of total amino acids to glutamine (TAA/Gln), essential amino acids to glutamine (EAA/Gln), glutamine to glucose (Gln/Glc), and oxygen to glucose (OUR/Glc), are also considered. A comparison of a number of cell lines including hybridoma, BHK, and CHO cells under a wide range of experimental conditions revealed that all the cell lines have similar patterns of variation of stoichiometry. In steady states of continuous culture, Lac/Glc and Gln/Glc are primarily determined by the residual glucose concentration while TAA/Gln and EAA/Gln correlate well with the residual glutamine concentration. Ammonium formation not only is a function of glutamine concentration but also is affected by the consumption of other amino acids, particularly at low residual glutamine concentrations. NH4+/TAA turned out to be a more suitable parameter to describe the ammonium formation. Large variations of all these stoichiometric ratios are found under conditions of relatively low residual concentrations of glucose and glutamine (both ca. < 0.2-0.5 mM). Above these concentrations the stoichiometric ratios are relatively constant and are independent of the cell lines. Thus, the correlations for these stoichiometric ratios may be directly used to control the nutrient concentration at low levels which are otherwise on-line difficult to determine. A stoichiometric equation is also derived for oxygen consumption. It is found that the metabolism of amino acids can significantly contribute to the consumption of oxygen. A correlation is obtained for OUR/Glc which may be used for the monitoring and control of mammalian cell cultures.

Ammonia↗

PubMedDate not supplied

Model simulation and analysis of perfusion culture of mammalian cells at high cell density.

Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.

Animals↗