Roller bottle

REASON FOR SUBMISSION This section should contain a brief explanation of the submission, e.g., "manufacturing change from roller bottle to cell factory" or "response to Information Request Letter of 1/9/97" or "Pediatric exclusivity determination request" or "to satisfy a subpart H postmarketing commitment". 

Special reactors are required to conduct biochemical reactions for the transformation and production of chemical and biological substances involving the use of biocatalysts (enzymes, immobilised enzymes, microorganisms, plant and animal cells). These bioreactors have to be designed so that the enzymes or living organisms can be used under defined, optimal conditions. The bioreactors which are mainly used on laboratory scale and industrially are roller bottles, shake flasks, stirred tanks and bubble columns (see Table 1). 

Whereas roller bottles and shake flasks are used for screening tests or for the cultivation of precultures, on production scale mainly stirred tanks, bubble columns, and in a few cases, also loop reactors are used (see Table 5). The stress in these reactors is therefore also of special significance and should be given particular consideration. 

As is the case with pure bubble columns and gas-operated loop reactors, most bioreactors in technical use are aerated with oxygen or air. Reactors with pure surface aeration, such as roller bottles, shake flasks and small stirred reactors or special reactors with membrane aeration, are exceptions. The latter are used for the cultivation of cells and organisms which are particularly sensitive to shearing (see e. g. [28 - 29]). The influence of gas bubbles in increasing stress has been described in many publications (see e.g. [4, 27, 29, 30]). In principle it can be caused by the following processes ... 

HeLa S3 (ATCC CCL-2.2) cells, a clonal derivative of the parent HeLa line (ATCC CCL-2), which are adapted to grow in suspension and therefore more suitable for large biomass production, are used for the preparation of human cell extract. The cells are maintained in suspension culture in Coming 850 cm2 Polystyrene Roller Bottles at 37° at a concentration of 3 to 6 x 105 cells/ml in Eagle s Minimum Essential Medium Joklik Modification (Sigma) supplemented with 10% Fetal Bovine Serum (Invitrogen) in the presence of 5% C02. 

For these proteins, mammalian cells proved better hosts, as they could process the protein with intracellular machinery similar to that in humans. However, large-scale production of proteins in cell culture was problematic. Mammalian cells had to grow attached to a solid surface, such as glass in roller bottle culture. While the Federal Drug Administration (FDA) had approved some processes for vaccine production that used cell cultures, it required that these cells be normal. Normal mammalian cells can divide only a few times before they stop growing, making scale-up to large volumes difficult. 

Different animal cell types display different properties pertinent to their successful culture. Those used to manufacture biopharmaceuticals are invariably continuous (transformed) cell lines. Such cells will grow relatively vigorously and easily in submerged culture systems, be they roller bottle or bioreactor based. 

Automated roller bottle cell culture systems, 5 35 It... 

The trypsinized cultures are counted and a sample is assessed for survival as for the cytotoxicity assay. In addition, an appropriate number of cells are reseeded for estimation of mutation frequency at the day 8 expression time. The cells are transferred to roller bottles (usually 490 cm2) for this stage. The bottles are gassed with pure CO2, the tops are tightened and the bottles are incubated at 37°C on a roller machine (approximate speed 0.5-1.0 rev min-1). Usually 106 7 8 9 viable cells are reseeded in 50 ml of Eagle s medium containing serum, but more cells are required at the toxic dose levels. 

The bottles are subcultured as necessary throughout the expression period to maintain subconfluency. This involves retrypsinization and determining the cell titre for each treatment. For each culture a fresh roller bottle is reseeded with a minimum of 106 cells. 

For transfection in roller bottles the protocol is scaled up as follows For each roller bottle (2125 cm ) 0.5 mg DNA is diluted with 50 ml serum-free DMEM and 1 ml PEI added. Following incubation at room temperature for 20 min,... 

Commercial scale cultivation of mammalian cells is accompHshed using different technologies roller bottles, microcarriers, suspension (batch, fed-batch or perfusion mode) and hollow fiber bioreactors (Table 2). However, especially for products needed in large amounts, suspension cultivation seems to be the most effective system [4, 5]. Suspension-based systems are characterized by a homogeneous concentration of cells, nutrients, metabolites and product, thereby facilitating scale-up [6] and enabling an accurate monitoring and control of the culture [7]. 

Cultivation method Fermentation Fermentation Fermentation (suspension cells) Roller bottle (adherence cells)... 

Cell inoculum is added to a defined volume of medium in either fermenter or roller bottles, and allowed to grow and Roller bottle be harvested at predetermined optimal time point... 

Similar in principle to chemostat configuration except the cells are retained within the fermenter and roller bottles using inline or external cell separation devices. The hollow fiber filter allows continuous separation of cells from tissue culture fluid containing products. In this configuration, the recombinant product is often designed to be excreted into medium allowing it to be collected in perfusate. 

Fermentation and alternative production techniques, such as roller bottles, can be carried out in four different ways. They are (1) batch process, (2) fed-batch process, (3) chemostat process, and (4) perfusion process. Batch and fed-batch processes require termination of cell growth while chemostat and perfusion processes allow continuous cell cultivation. 

In a batch configuration, host cells that contain an expression vector for the recombinant product are added to a predetermined volume of growth medium (Figure 4.12A). The cells are allowed to grow until the nutrients in the medium are depleted or the excreted by-products reach inhibitory levels. At that time, the cells are harvested, and recombinant protein, found in inclusion bodies, cell-membrane fractions, or cytoplasm, are isolated after disruption of the harvested cells. Because the host cells are destroyed at the end of each run, they must be replaced every three to seven days for fermentation or every two weeks for roller-bottle or microcarrier-support production of adherent cells. To ensure uniformity and reproducibility, the FDA requires manufacturers of recombinant proteins to establish and validate a seed stock of recombinant host cells that are validated to contain the characterized expression vector and to be free of contaminants. 

Epogen (Epo) Stimulation of erithropoiesis rCHO, Roller bottles... 

For the cultivation of animal cells, inoculum expansions have traditionally been conducted in T-flasks, shake flasks, spinner flasks, or roller bottles. Typi-... 

Depending upon the design of an experiment, cells were grown in 60 mm petri dishes, 150 cm2 plastic flasks (Falcon no. 3024), or roller bottles in MEM supplemented with 12% fetal bovine serum and antibiotics in 95% air, 5% C02 water humidified incubator at 37°. All experiments were initiated with confluent mono-layer of cells grown for about 5-15 generations. 

Approximately 2.5 x 10 each of normal (B.P.) and FH receptor negative homozygous fibroblasts (T.B.) were seeded into roller bottles and grown as described above. 

Fermentation can only be used for cells that can grow in suspension. This includes most prokaryotes (including E. coli) and lower eukaryotes (including yeast) but only a small percentage of mammalian cells. Most recombinant mammalian cells require a surface support to replicate. These cells, known as adherence cells, are typically grown in roller bottles. 

The range of culture flasks and reactor types employed is quite wide, both for suspension and adherent cultures, from small Carrel s or Roux s flasks to roller bottles. Fixed- and fluidized-bed bioreactors, air-lift reactors and even stirred and aerated tanks with capacities up to 15 m3 are common in large plants producing monoclonal antibodies (mAbs) for anticancer therapies (Adams and Weiner, 2005 Griffiths, 1988). 

Cryotube Stationary flask Roller bottles Production bioreactor... 

Stirred-tank bioreactors Air-lift bioreactors Wave bioreactors Microcarrier-based systems Packed-bed bioreactors Fluidized-bed bioreactors Hollow-fiber bioreactors Bioreactors providing surfaces for attached cell growth (roller bottles, CellCube , Cell Factory)... 

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