Claus Emborg

Extra- and intracellular amino acid concentrations in continuous Chinese hamster ovary cell culture.

A recombinant DNA Chinese hamster ovary (CHO) cell line that produces tissue-type plasminogen activator (tPA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day with three different asparagine concentrations in the feed (0.05, 2.55 and 7.55 mm). The up-shift in asparagine concentration caused an up-shift in asparagine consumption [15.7 and 31.4 nmol (106 cells)−1 h−1] and intracellular concentration (2.19 and 18.7 mm). The up-shift was accompanied by an increased production of ammonium, glycine and alanine, and a metabolic shift whereby the cells began to produce aspartate and glutamate, which were consumed before the shift. The tPA production was reduced in the up-shift culture. This might be explained by ammonium inhibition, but alternatively by a surprising down-shift in the intracellular concentration of many amino acids, a down-shift that was not observed in the extracellular concentrations or consumption rates. For efficient physiological engineering of mammalian cells it is necessary to include both extracellular and intracellular measurements and to consider the transport into and out of the cells.

Modeling the growth and proteinase A production in continuous cultures of recombinant Saccharomyces cerevisiae

Overexpression of the homologous protein proteinase A (PrA) in Saccharomyces cerevisiae has been achieved by inserting the PrA gene (PEP4) with its own promoter on a 2mu multicopy plasmid. With this system the specific PrA production rate was found to be described well by a linear function of the oxidative glucose metabolism, the reductive glucose metabolism, and the oxidative ethanol metabolism, with a significant lower yield resulting from the reductive glucose metabolism compared with the oxidative glucose metabolism. To describe the experimental data, a simple mathematical model has been set up. The model is based on an assumption of a limited respiratory capacity as suggested by Sonnleitner and Käppeli but extended to describe production of an extracellular protein. The model predicts correctly the critical dilution rate to be between 0.15 and 0.16 h(-1), the decrease in the biomass yield above the critical dilution rate, and the production of proteinase A at different dilution rates. Both the experimental data and model simulations suggest that the optimum operating conditions for protein production is just at the critical dilution rate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 447-454, 1997.

Enhanced antibody production associated with altered amino acid metabolism in a hybridoma high-density perfusion culture established by gravity separation

A high density hybridoma perfusion culture was established by separating and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator — an inclined modified Erlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day−1 had been reached, viable cell density stabilized at around 10×106 cells ml−1, a level five times that obtained by simple batch culture. The efficiency of the separator was enhanced by cell flocculation. Specific antibody productivity, which was initially 0.4 μg 1×106 cells−1 h−1, decreased to half that value while cell density was increasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viable cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important substrates for energy generation, glucose and glutamine, caused their broth concentrations to decrease to 1.5 mM and 1 mM, respectively, from input medium concentrations of 25 mM and 10 mM, respectively. At the same time there was an increase in the specific production of glycine and aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, respectively. We suggest that this shift in metabolism results in enhanced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more energy efficient metabolism. The mechanism whereby this leads to enhanced specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtained with the present perfusion culture resulted in a high monoclonal antibody production −100 mg l−1 d−1.

Bacillus sphaericus V-penicillin acylase I. Fermentation

SummaryIn an aerobic fermentor with a fermentation medium based on 4% corn steep liquor and salts, Bacillus sphaericus, strain NCTC 10338, produces an intracellular V-penicillin acylase, when pH is in the narrow interval 7.5–8.0. The strain grows well outside this pH range, but the growth is not associated with acylase activity in the cells. The enzyme was not induced by phenoxy acetic acid and was not repressed by glucose.

Observations on the influence of glutamine, asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells

When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.

The influence of acetic acid on penicillin production

SummaryFed-batch fermentations with Penicillum chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and acetic acid. When 20% of the carbon source was acetic acid, yields of penicillin-V were 25% higher than in fermentations where glucose was the only carbon source in the feed. The increased yield was due to higher specific productivity and/or cell mass. The effect was seen in fermentations where the carbon source was fed at a constant rate and the pH kept automatically at 6.5 by addition of inorganic acid or base, as well as in fermentations where pH controlled the addition of feed.

Conversion of cephalosporin-c into 7-phenoxy-acetamido-cephalosporanic acid by acyltransferase of mutants of Penicillium chrysogenum

SummaryWe have tested the acyl-transferase of penicillin producing strains and penicillin-less mutants of Penicillium chrysogenum for their ability to exchange the d-α-aminoadipic acid side chain of cephalosporin-C with phenoxyacetic acid. We found the reaction in both types of strains.

Production of monoclonal antibodies against the enzyme nuclease fromSerratia marcescens and evaluation of the individual antibodies for their use in EIA and in affinity chromatography

Seven different monoclonal antibodies against the enzyme nuclease were compared with respect to their binding strength and to elution from EIA plates coated with antigen by ten different desorbing reagents. The combination of affinity ranking, obtained by specific titers, and results from desorption with chaotropic reagents and MgCl2 serve as criteria for classifying the antibodies for use in EIA assays or for affinity chromatography. The introduction of an “elution assay” early during the screening of new hybridomas can lead to an early evaluation of parameters which are important for the subsequent use of monoclonal antibodies. Such a procedure will therefore save both costs and efforts.

An evaluation of fed-batch cultivation methods for mammalian cells based on model simulations

Fed-batch cultivations of hybridoma cell cultures for antibody production were computer simulated by a detailed structured model, that divides the cell mass into four metabolic compartments and makes use of descriptions of the most important metabolic pathways. Surprisingly the simulations indicate, that online or frequent off-line control of fed-batch hybridoma cultures may lead to low and variable yields of antibody product. With less frequently controlled fed-batch cultures the system is more stable; and there is an optimal time interval for control, that depends on the feeds selected. The product yields in optimal fed-batch cultivations, however, are seldom higher than in comparable batch cultivations.

Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: Experiments and computer simulations

Overproduction of proteinase A by recombinantSaccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor. Memebrane filtration was used to separate cells from the broth. Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both experimentally and by computer simulations. Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling. Additional simulations showed that the proteinase A concentration decreased drastically at high dilution rates and the optimal volumetric productivities were at high dilution rates just below washout and at high ratios of recycling. Cell-recycling fermentation gave much higher volumetric productivities and stable product concentrations in contrast to simple continuous fermentation.