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)     

Metabolic enhancement due to plasmid maintenance

Summary Plasmid maintenance allows the strain JM109 of Escherichia coli to grow in a minimal defined medium (M9). JM109 carrying no plasmid can hardly grow in M9 whereas JM109 carrying one, two and three plasmids have a clear metabolic advantage over the untransformed strain. In a complex medium like LB (Luria-Bertani Broth) all strains grow well and despite the number of plasmids carried by the host maximum specific growth rates are not severely affected. Our results suggest that the glucose metabolism is an essential factor contributing to this behavior.     

Mathematical simulation of the growth of a three plasmid harboring Escherichia coli JM109 strain and the production of the fusion protein EcoRI::SPA with a four-compartment model

In the previous papers [35, 36] the process variables of plasmid-free, one-, two- and three-plasmid harboring E. coli JM109 cells were investigated in batch and continuous cultivation as a function of the medium composition, plasmid content, dilution rate and cultivation (generation) time. In the present paper the growth of the recombinant E. coli JM109 [pEcoR4, pRK248cI, pMTC48] and the production of the fusion protein EcoRI::SPA are simulated by using a four-compartment model, consisting of the active cell components (ribosomes, mRNA, tRNA, and others) (A), the structure forming materials and chromosomal DNA (Z), the plasmid-DNA (G) and the recombinant enzyme protein (E). At the first time, all of the three plasmids: the production plasmid (Gp), the repressor plasmid (Gr) and the protection plasmid (Gs) are taken into account in the plasmid DNA-compartment of the model. The calculated and measured courses of the cell mass, the concentrations of glucose and acetate, and the products as well as the particular plasmids agree well.     

Influence of age and structure of Pencillium chrysogenum pellets on the internal concentration profiles

Pellets of Penicillium chrysogenum which were spontaneously formed after a certain stage of a batch fermentation, displayed a considerable structural change in course of their lifetime. Microelectrode studies showed the internal mass transport properties of these pellets (diameter 1–3 mm) to be highly effected by their morphological structure. Relatively young pellets, in an early stage of the batch fermentation, possessed a homogeneous and dense structure. These pellets were only partly penetrated by oxygen (ca. 70 μm) at air saturated bulk conditions. Older pellets, in a final stage of the batch fermentation, were stratified and fluffy. They were completely penetrated by oxygen due to a decreased activity and a higher diffusivity. Investigations with glucose microelectrodes revealed that glucose consumption inside pellets of all lifetimes exclusively occurred in the periphery, indicating that growth was restricted to these regions only.     

The biomechanical relation between incisal and condylar guidance in man

The arrangement, form and movement of incisal and condylar guidance allow representation of the stomatognathic system by a gearing with positive drive and closed linkage. Because of problems of stability, this gearing has to be a throttle crank. Therefore, the stomatognathic elements have to fulfill the condition of Grashoff. The analysis of 14 Class I patient data confirms this condition and yields that their stomatognathic systems seem to be determined by only two parameters. The properties of these systems are largely independent of the jaw's growth if the momentary growth takes place in the direction of the initial inclination of hinge-axis movement out of centric relation.     

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.     

Immobilized α-chymotrypsin: Pore diffusion control owing to pH gradients in the catalyst particles

The fast enzymatic hydrolysis of D ,L -phenylalanine methylester (DLE) to L -phenylalanine (LA) and D -phenylalanine methylester (DE) with immobilized α-chymotrypsin was chosen as a model reaction. Under the experimental conditions applied in the present investigations the pore diffusion is the rate-limiting step of this reaction owing to the pH gradient in the particles. The effectiveness of the catalyst is experimentally determined as a function of the substrate concentration based on measurements of the enzyme protein content of native and immobilized enzyme. The proteolytic reaction is theoretically treated by also using a pore diffusion model which takes into account the concentration gradients of substrate and product, pH- and enzyme activity profiles, as well as the change of buffer capacity of the solute in the catalyst particles. The model parameters were experimentally determined for the investigated system. It can be shown that conditions are possible for which the effectiveness of the catalyst exceeds unity.     

Comparison of the continuous flotation performances of Saccharomyces cerevisiae LBG H620 and DSM 2155 strains

Saccharomyces cerevisiae LBG H620 and DSM 2155 strains were continuously cultivated under carbon (C)-limited, phosphorus (P)-limited and nitrogen (N)-limited growth conditions. Cell and protein concentrations in feed, foam, and residue as well as the degree of cell recovery and the rate of foaming were measured, and the concentration and enrichment factors were evaluated at different dilution rates (D). The LBG H620 cells were reduced, while the DSM 2155 cells were enriched in the foam. The highest concentration factors in DSM 2155 cells were attained if they were cultivated under strong P-limitation at a low D. Fairly high concentration factors were also found under C-limitation. Under N-limitation, low concentration factors were found with low Ds. At the beginning of the continuous cultivations, all of the cells were recovered, but with advancing time the degree of recovery and cell concentration and the enrichment factor ratio diminished. The cellular properties of the yeast were characterized by flow cytometry, and the surface properties by measurements of their hydrophobicity, electrophoretic mobility, and chemical composition (using X-ray photoelectron spectroscopy, XPS). These investigations indicated that the large difference in flotation between the two strains is due to different surface properties. Strain DSM 2155 has higher surface hydrophobicity and lower electrokinetic potential. Cell wall properties and the cell flotation depend on medium composition and age of the culture.Correspondence to: K. Schügerl