Page 156 - 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

Mammalian Cell Culture 41

multiple nozzle injection on the medium surface, caged 1. Hollow-Fiber Bioreactors
aerator, spin exchange aerator, external and internal loop
This method was pioneered by Knazek in 1972 using ul-
oxygenator, and membrane tubing arranged in the ves-
trafiltration capillary fibers. They can be considered anal-
sel or stirring system. The use of closed perfusion loops
ogous to a blood vascular system as the fibers selectively
through external reservoirs is also an efficient means of
(by molecular weight cut-off) allow passage of macro-
oxygenation as long as the perfusion rate is fast enough,
molecules through the spongy fiber wall (60 µm) as the
which requires a very efficient spin filter or other cell sep-
medium flows continuously through the lumen (200-µm
aration device in the culture.
diameter). Cells are kept in the extra capillary space. A
unit consists of thousands of fibers “potted” at either end
3. Large-Scale Bioreactors in a cylindrical housing and capable of supporting both
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Large-scale bioreactors are based on either the fermenter ADC and suspension cells at 1–2 × 10 /mL. This con-
tank or the airlift principle. Stirred tanks have been oper- cept has been widely developed with many commercial
ated at 8 to 10,000 L for the production of interferon, tPA, units available and has been used particularly for produc-
recombinant antibodies, and DNase, and airlift at 2000 L ing monoclonal antibodies. The principal limiting factor
principally for monoclonal antibodies. The preference is in their exploitation is the difficulty in scaling-up beyond
for tank fermenters, as they are a well-tried and familiar very small volumes (25 mL). Thus, this method gives a
production method using conventional production plant very large surface-to-volume ratio, allows continuous re-
facilitiesandhavebeenrefinedoverthepast30years;how- moval of waste products and supply of nutrients, supports
ever, most processes run at scales between 50 and 500 L. high cell densities with a tissue-like architecture, and al-
Containment of the process to prevent ingress of con- lows a concentrated product to be harvested. The disad-
taminating factors and the release of materials that may vantages are diffusional limitations causing culture inho-
affect the process worker and environment is a key factor mogeneity and cell necrosis, process control complexity,
in these processes. Microbiological containment of small- and difficulty in sterilization.
scale systems (below 20 L) in class III microbiological
cabinets is possible, and a larger scale fermentation plant
2. Spin Filters
can be contained to P3 standards at least up to the 150-L
scale. Asallstaticfilterswithinaculturebecomeblocked,sooner
The engineering complexity and considerable resource rather than later, the development of a rotating filter that
investment in money and time to get a large-scale pro- creates a boundary effect, thus delaying cell attachment
duction process built and operating mean that many new and filter clogging, has been an important step forward.
products are still brought to market using replicate small- These filters provide a simple technical solution to scaling-
scale systems. This allows a quicker market entry, but it up process intensity within well-established stirred fer-
does involve a complete re-registration of the product if, menters. They are limited in that clogging does eventually
at a later stage, the product is moved to a large-scale pro- occur and in the perfusion rate that can be maintained.
duction process. However they are particularly useful for microcarriers, as
a far larger mesh can be used, thus reducing the onset of
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filter clogging and permitting cell densities of 2 × 10 /mL
C. High-Cell-Density Bioreactors to be attained.
In this section, the processes that support cells at signif-
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icantly greater densities than the classical 2–3 × 10 /mL
3. Microporous Microcarriers
level are described. In the human body, cells in tissues are
9
3
found in the order of 2–3 × 10 cells/cm , and this propor- Microcarrier culture has proven to be the most effective
tion has always been a target for in vitro systems. However, scale-up method for ADC, despite its limitations (critical
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2 × 10 cells/mL have so far been the practical limit of procedures, low surface area-to-volume ratio of a sphere).
density scale-up, but even so this is a 100-fold increase. To To increase the surface area porous particles were devel-
achieve higher unit cell density requires (1) perfusion to oped. The initial particle was the Verax microsphere which
supply nutrients and remove waste products, and (2) cell was 500 µm in diameter and manufactured from bovine
immobilization in order to perfuse media at a fast enough collagen. The interconnecting channels of 20- to 40-µm
rate without damaging or washing the cells out of the diameter provided an internal open volume of 80% of the
culture. A huge range of devices have been developed to sphere. The spheres were fluidized at 75 cm/min upward
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meet these requirements, of which hollow-fiber bioreac- flow, and cell densities in excess of 10 /mL intrasphere
7
tors, spin-filter perfusion, and microporous microcarriers volume were achieved (equivalent to 4 × 10 /mL in the
are the most successful in terms of acceptance and use. bioreactor). The sphere matrix provided a huge surface