G. Blüml

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.

Large-scale mammalian cell culture.

Over the past year, progress has been made toward a better understanding of the physiological and biochemical responses of cell cultures to various environmental changes. Much of the theoretical and experimental work that has been reported will help improve expression in mammalian cell culture, which is one of the key steps in biopharmaceutical manufacturing.

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.

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).

Process Development for the Large Scale Production of Clinical Grade Human Monoclonal Antibody Using a Cytopilot Fluidized Bed Reactor

Monoclonal antibodies for passive immunotherapy demand for cheap large scale manufacturing technologies. Previous studies showed that fluidized bed reactors (FBR) are a promising production system considering scale up potential and volumetric productivity. r-CHO cells were cultivated on 200–400 ml settled volume of macroporous microcarriers (Cytoline 1, Pharmacia Biotech) in a Cytopilot-Mini FBR to optimize relevant parameters for large scale production of clinical grade material in a 120 1 FBR (20–301 of packed bed). Main focus was put on i) balancing medium composition to enable reasonable perfusion rates, ii) optimized carrier preparation, iii) high efficiency of cell attachment during inoculation iv) fast change from serum supplemented propagation medium to protein free production medium.

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^.^-^