T. Gaida

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.

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-termin 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 extendedin 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.

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 108 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.

Long-Term Stability Of Continuously Perfused Animal Cells Immobilized On Novel Macroporous Microcarriers

The ultimate goal of optimizing biological production is the establishment of simple, safe, cheap, consistent, and easily manageable production systems. Various complicated techniques have been used for animal cell culture. In practice, however, only the simple ones are used for production purposes. A high cell density continuous perfusion in vitro system simulates a situation that most closely exists in the in vivo maintenance of differentiated cells arrested in the G1/G0-phase of the cell cycle. In order to make this a technological reality, a macroporous open structured carrier has been designed using high pressure polyethylene as a primary matrix material. During long-standing continuous perfusion of cultured mammalian cells such as Chinese hamster ovary (CHO) cells with a protein-free medium, the cells are maintained at high cell density (> 10 8 per ml) in a fully productive but nonpropagating state. Using the fluidized bed porous bead technology we do not expect any limitation in the scale-up of any technology using continuous mammalian cell lines.

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.

PRODUCTION OF THE HIV-1 NEUTRALISING HUMAN MONOCLONAL ANTIBODY 2F5: STIRRED TANK VERSUS FLUIDIZED BED CULTURE

ABSTRACT The performance of a 6 litre stirred tank reactor and a fluidized bed reactor operated with 2 litre macroporous PolyporE beads have been compared. Under controlled perfusion conditions the productivity (anti-HIV-1 lgG3) of the fluidized bed culture system turned out to be 20-fold higher.

Monoclonal Antibody Production Using the Porous Glass Bead Immobilization Technique

The increase of cell density in bioreactors and thus immobilization techniques have received a substantial amount of attention.s2 The technique for mass production and maintenance culture of anchorage-dependent mammalian cells at high cell densities is established for the production of a wide range of biological^.^^^ There are a number of reasons claimed beneficial for using immobilization techniques for the propagation of hybridomas, for example, high cell density, decreased biomass separation cost, lower media costs, the use of protein-free media, and higher volumetric production rate^.^-^

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.

OXYGENATION IN FLUIDIZED BED BIOREACTORS USING THE MICROSPARGING TECHNIQUE

ABSTRACT An oxygenation system based on a microsparger with a stainless steel mesh with a pore size of 0.5 micron for oxygen supply in high density fluidized bed bioreactors is described. The design of cell culture processes can be improved with respect to sterility and simplicity. The microsparger applied in the 80 liter pilot scale fluidized bed bioreactor has an oxygen transfer capacity of 5 000 mg O2/h. Under standard conditions one cm2 of the microsparger can support 1012 cells.

HIGH DENSITY AGGREGATE CULTURE OF RECOMBINANT CHO CELLS IN FLUIDIZED BED BIOREACTORS

ABSTRACT The propagation of anchorage-dependent CHO cells as cell aggregates in a bench scale fluidized bed bioreactor system is shown. The aggregation of the cells gives diameters of up to 4 000 μm having reasonable sedimentation velocity for fluidization and retention. The achieved cell densities were in the range of 2–3×10 8 cells/ml settled aggregate volume. Maximum cell number and volumetric productivity showed a 5-fold increase compared to the initial culture, whilst specific glucose consumption was nearly unchanged.