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Biomedical subjects

T Gaida

Publications and source records attributed to T Gaida.

10 recordsLinked to original sources
PubMed1994

Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems.

A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates. In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis. The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining. The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates. The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant. Hardly any alterations in two-dimensional pattern were detectable. The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.

Animals↗
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↗
PubMed1992

Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads.

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.

Animals↗
PubMed1992

Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry.

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.

Animals↗
PubMed1992

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology.

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-term in vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extended in vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Animals↗
PubMed1991

Modular integrated fluidized bed bioreactor technology.

We describe the design and demonstrate the application of a modular integrated fluidized bed bioreactor system. Basically the system is a reactor vessel equipped with an extending cylinder and a liquid distributor plate. Instead of an external recirculation loop, as used in existing fluidized bed systems, a low shear stress impeller is used as the recirculation pump. The system has several unique features, such as modular exchangeable elements, efficient oxygenation and the option of operating as a stirred tank-, a packed bed- or a fluidized bed reactor. An example of a fluidized bed run using CHO-K1 cells is shown. Under standard culture conditions a 100-fold increase in cell density (up to 1.2 x 10(8) cells/ml) was achieved.

Animals↗
PubMed1990

The use of macroporous gelatin carriers for the cultivation of mammalian cells in fluidised bed reactors.

A fluidised bed system for the cultivation of mammalian cells on a new type of macroporous gelatin microcarrier is described. The volumetric cell densities achieved under controlled conditions for two 'standard cell lines' (VERO, CHO) were one order of magnitude higher compared to conventional techniques using spherical microcarriers. The system can be potentially used for both anchorage dependent and independent cells.

Animals↗
PubMedDate not supplied

A novel ultrasonic resonance field device for the retention of animal cells.

This article describes two types of flow-through cell retention devices based on the concept of layered piezoelectric resonators. A single-chamber device is compared to a novel optimized steam-sterilizable prototype ultrasonic cell separator with improved acoustic design and an integrated cooling circuit, eliminating the problem of local temperature increase caused by the high amplitudes necessary to achieve the separation of animal cells with low acoustic contrast. This setup yields highly reproducible results and is ideal for studying the long-term effects of ultrasonic sound fields and separation efficiency. The novel two-chamber system has the potential for scaleability due to the reduction in thermal and acoustic flow, increased field stability, and separation efficiency. Finally, the effect of power input on separation and cell viability is reported. Such flow-through cell retention systems could be used as systems to retain biomass within the fermentor or as a substitute for centrifugation, with the major advantage of eliminating high-speed rotational motion.

Animals↗
PubMedDate not supplied

Selective retention of viable cells in ultrasonic resonance field devices.

A double-chamber ultrasonic resonance field device was used for the separation and retention of animal cells. By controlling operational parameters such as flow and power input, the device can retain viable cells more efficiently, allowing for selective removal of nonviable cells and cell debris. A simple model describing the forces acting on spherical particles in a sound field (primary radiation force, Bernoulli force, secondary radiation force) is presented. Field stability increases with decreasing average flow rates and increasing power input. At very high field stability, as achieved with low flow rates and high power input, the selectivity for viable cells is reduced, due to the efficient retention of all types of particles. At high flow rates and resulting low field stability, selectivity is also reduced, due to poor separation efficiency, resulting in equally low retention of viable cells, nonviable cells, and cell debris.

Animals↗