Page 157 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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Encyclopedia of Physical Science and Technology EN009G-399 July 6, 2001 20:4

42 Mammalian Cell Culture

area for attachment of ADC, but was equally suitable for only truly scalable high-cell-density system available, and
suspension cells entrapped in the pores. The system was provides a micro-environment that stimulates cell product
scaled up to 24 L routinely and to 200 L in the company’s expression.
production unit. It was used for over 85 different cell lines
producing many cell products (including tPA, proUK,
EPO, IFN, IL, factor VIII, various immunoglobulins). IV. THE CULTURE PRODUCTION
Many microporous beads are available, as are systems PROCESS
such as the Pharmacia Cytopilot using Cytoline porous
microcarriers. There is a preference for microporous mi- The basic principles common to most processes are sum-
crocarriers that could be used in stirred, rather than flu- marized in Fig. 4. The components are
idized, bioreactors, and these are now available (Cellsnow
and ImmobaSil). ImmobaSil is of particular value as it is 1. Seed banks: Both the cell line and virus for vaccines
extremely permeable to oxygen, is a non-animal product have to be laid down in a fully tested and characterized
safe from bovine contaminants, is robust, and can be used bank. Thus, each production batch will be initiated from
in all culture modes. identical cells (and virus) known to be viable and contam-
An alternative to using stirred/fluidized microporous ination free.
carriers is to use a fixed bed of porous glass spheres (e.g., 2. Cell seed expansion: A series of culture steps is
Siran). Fixed bed reactors of solid glass spheres have many needed to expand the cell seed ampoule (e.g., 5 million
9
advantages, and the disadvantage of low cell density can cells) to production size (range 10 to 10 13 cells). For
be overcome by using 5-mm Siran spheres. These have HDC, this is accomplished in steps of a split level of 1:2
2
a surface area of 75 m /L with interconnecting pores and or more usually 1:4 through a series of flasks, roller bot-
channels of 60 to 300 µm. They can be stacked in 5-L beds tles, and possibly cell factories (A/S Nunc). Other cell
and perfused at 5 linear cm/min to provide a high feed rate types are split at a 1:5 to 1:20 ratio. A similar build-up is
without washing out or damaging the cells (protected from needed for the virus seed.
shear within the particles). The versatility and usefulness 3. Production: The production culture may be a batch
of this technique have been demonstrated for a range of of several hundred roller bottles, 30 to 50 cell factories, or
suspension and anchorage-dependent cells, resulting in a a single bioreactor for suspension (100 to 10,000 L) or
tenfold higher productivity over equivalent systems. microcarrier (50 to 500 L) cells. Although batch-type
A characteristic of all microporous carrier systems is production is still the most common process, continuous
that cell-specific productivity is always higher, presum- processes where the product is harvested daily over a long
ably due to the favorable environment of cells packed to- period (20 to 100 d) are being increasingly used. Cul-
gether in almost tissue-like density. Commercial fixed- ture systems based on hollow fibers, porous microcarriers,
bed reactors are available (Meredos GmbH, D-37120 or other immobilization techniques are used for contin-
Bovenden). uous perfusion processes. During the production phase,
The advantages of porous carrier culture are summa- the virus seed or a promoter (e.g., for interferon) may be
rized in Table III. This technology is truly universal as it added.
is equally suitable for suspension and ADC, is flexible in 4. Harvesting: If the product is intracellular, then the
the range of bioreactor systems it can be used in, is the cells have to be harvested (trypsin and/or EDTA), washed,
and concentrated by centrifugation. Extracellular (se-
TABLE III Advantage of Microporous Microcarriers creted) products just require the collection of the culture
1. Unit cell density 50- to 100-fold higher than free suspension supernatant.
2. Suitable for both attached and suspension cells 5. Downstream processing: Intracellular products have
to be extracted from the cells (by sonication, freeze
3. Can be used in fluidized and fixed-bed reactors and in stirred
suspension thawing, and/or homogenization), and separated from
4. Elimination of seed chain steps by in situ 100- to 250- fold seed the cells (centrifugation or filtration). Extracellular prod-
expansion ucts require concentration and separation from the bulk
5. Efficient diffusion into a sphere (30% diameter penetration = 70% supernatant.
of volume)
6. Formulation: The product is added to a medium
6. Protection of cells from shear
with protective and stabilizing agents and then usually
7. Three-to fivefold increase in specific cell productivity
freeze-dried.
8. Easily derivitized three-dimensional structure for specialized cells
7. Quality control: Throughout the process, prescribed
9. Capable of long term (>100 d) culture with continuous harvesting
samples are taken for a range of quality control tests to
10. Scale-up potential as compared with analogous systems
show safety, efficacy, and consistency in the process and
(microcarrier to 4000 L)
product.