N. Zach

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.

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.

SCALE-UP OF A FLUIDIZED BED REACTOR OPERATED WITH POROUS GLASS CARRIERS

ABSTRACT The IAM modular fluidized bed reactor was scaled up from laboratory size (7 1) to pilot scale (80 1). Hardeware was designed to fit the modular IAM pilot plant structure and software was adapted to the new reactor design. The new reactor was tested using porous glass beads and CHO cells as model cell line. After inoculation and batch phase a continuous perfusion system using protein free-medium was set up and operated over a period of three weeks. At the final dilution rate (approx. 12 times the carrier volume) a cell density up to 5 × 10 7 cells/ml carrier bed could be achieved. Fermentation control was achieved using an industrial direct digital process control system (Honeywell TDC 3000).

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^.^-^

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.

A SIMPLE MAGNETIC DRIVEN MINI-REACTOR FOR FLUIDIZED AND PACKED BEDS

KEYWORDS: fluidized bed, packed bed, labratory bioreactor, mammalian cells, macroporous carriers

ON-LINE GLUCOSE CONTROL OF ANIMAL CELL CULTURES IN FLUIDIZED BEDS

ABSTRACT High cell density fermentation systems (e.g. using micorcarriers) result in high volumetric substrate conversion. Control of metabolite concentrations is essential to prevent nutrient limitations or toxic levels of catabolites. In this work Glucose was automatically controlled at a constant concentration level. For aseptic sampling an in house developed cross-flow filtration module was used. Measurement was done by a PC-managed FIA-Biosensor system. Commercially available as well as self made sensors were tested. Glucose values were periodically used for calculation of feeding rate. Due to the design (automated data validation, exchangeable sampling and sensing modules) the system can be applied to long term animal cell culture fermentation.