Module 2 – GROWTH OPERATIONS
A Practical Guide to Biopharmaceutical Manufacturing
CHAPTER 2
CULTURE TYPE
All types of micro-organisms are grown in suspension with the exception of cell culture, in which cells
can also be anchorage dependent.
•
Suspension – the micro-organism can grow successfully whilst in a suspension
•
Anchorage dependent – the micro-organism requires a surface on which to attach itself and
grow
Suspension based operation is favoured for large scale manufacture. Mammalian cells are naturally
anchorage dependent. In comparison with suspension cultures, anchorage dependent cells are usually
more genetically stable and have higher productivities. Modification of mammalian cell lines is possible
to move from attachment to suspension based operation using one of three different methods:
•
Modification of the growth medium to prevent attachment, which may reduce productivity
•
Selective use of cell lines to reduce attachment dependency
•
Selective use of cells that will not attach to specially treated surfaces
The second option is suitable for production cell lines, but it can be difficult to achieve. The third
option is normally easier and more rapidly developed, allows operation at large scale, and enables
attachment dependent growth at small scales, simplifying laboratory handling and development.
2.1
Suspension culture
Some micro-organisms grow freely suspended in culture fluid. Nutrients are supplied by the growth
medium in which the micro-organisms are suspended. Oxygen is supplied by pumping air into the
bioreactor. To support the high cell densities required for economic operation, the suspension must be
agitated to ensure consistent and effective mixing so that all of the cells have access to nutrients and
oxygen and that excess heat and exhaust gas are removed. The agitation method differentiates the
two basic types of suspension bioreactor:
•
Air lift – agitation supplied by the movement of air through bioreactor
•
Stirred tank – mechanical impellers agitate the suspension
For small volumes, it is possible to use alternatives such as shaker flasks, mixing bowls, and disposable
wave bioreactors.
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High cell densities in suspension cultures are achieved by
consistent and efficient agitation to ensure that all the cells
have access to the required nutrients and the removal of
excess heat and exhaust gas
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Module 2 – GROWTH OPERATIONS
A Practical Guide to Biopharmaceutical Manufacturing
Irrespective of the agitation method, bioreactors are designed to avoid the introduction of any
unwanted materials, or contaminants. This requires careful design of all connections, tubing, and the
vessel itself to prevent areas that cannot be properly cleaned and sterilised. Bioreactors and associated
pipework surfaces are usually constructed of stainless steel and finished to a high standard to reduce
attached growth and aid cleaning. Any entry or exit point from the vessel is a potential contamination
route. The overall risk of contamination is often reduced by running the entire vessel slightly above
atmospheric pressure, impeding the entry of external organisms.
Cell cultures produce heat, and the larger the working volume, the greater the heat load. The vessel is
therefore cooled during the fermentation process to ensure that the process operating temperature
remains at the optimum value. Cooling is often supplied by external cooling jackets or coils. Internal
coils may also be used to allow more rapid cooling of vessels following sterilisation. This may be of
greatest benefit if the process requires the sterilisation of growth media in situ.
2.1.1
Airlift bioreactors
Air-lift bioreactors achieve circulation of the growth medium and aeration by injecting air into the lower
part of a bioreactor vessel (see Figure 2.1). The movement of the air mixes the growth medium and
generates a re-circulating flow through the vessel as shown in Figure 2.1. The air reduces the density
of the broth and causes it to rise up the draught tube. Gases separate from the broth at the top of the
bioreactor and are drawn off, increasing the broth density. The broth then re-circulates down to the
base of the bioreactor. The transport of oxygen into, and waste gases out of, the bioreactor are
controlled by diffusion of the gases into and out of bubbles.
Figure 2.1: An airlift bioreactor
Gas Outlet
Liquid Level
Draught Tube
Air Sparger
Air-lift bioreactors typically have height to diameter (aspect) ratios of greater than 8:1, resulting in tall,
slender vessels to achieve sufficient mixing. When required, additional mixing may be introduced by
the use of stationary fittings that generate additional turbulence and break up bubbles. This increases
the surface area over which the air and growth media interact and so increases the rate of oxygen and
waste gas transfer.
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© Informa UK Ltd, 2006
Module 2 – GROWTH OPERATIONS
A Practical Guide to Biopharmaceutical Manufacturing
Air-lift bioreactor vessels have no moving parts and often little internal structure, which helps reduce
maintenance requirements and simplifies cleaning regimes. However, the efficacy of this method is
inversely proportional to the size of the vessel.
Depending on the characteristics of the broth, there tends to be an upper limit to the realisable scale
of operation before additional mechanical agitation is required for adequate oxygen transfer. The
largest air-lift bioreactor used for the manufacture of a single cell protein, by ICI, was 1.5M litres.
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2.1.2
Air-lift bioreactors achieve mixing of the growth medium and
aeration by injecting air into the lower part of a bioreactor
vessel
Stirred tank bioreactors
In contrast to air-lift bioreactors, stirred tank bioreactors (Figure 2.2) use mechanical devices to agitate
the growth medium. Impellers and baffles within the vessel cause turbulence and allow finer control of
the oxygen and mass transfer processes. Stirred tank systems are readily scaleable, and typically use a
more squat configuration, with aspect ratios of approximately 3:1 or less.
Figure 2.2: Stirred tank bioreactor
Motor
Gas Outlet
Foam
Breaker
Flat Blade
Impeller
Baffle
Air Inlet
Drain Valve
The internal configuration of the vessel has a direct impact on the efficiency of the mixing, and many
different designs exist. Typically the mixing is achieved using rotating impellers and fixed baffles
attached to the inside of the vessel. Four to six baffles approximately one tenth of the overall vessel
diameter are commonly used. Stirred-tank reactors generally contain a series of impellers.
A large number of different impeller designs exist and may be used in specific applications to generate
the required agitation. The choice of impeller design is determined by the micro-organism being
grown. Impellers can be either a combination or single use of the two main types:
•
Axial dispersion – designed very much like a ship propeller (in fact one of the first designs was
called the Marine Impeller), this type of impeller moves the fluid towards the top of the vessel
•
Radial flow – moves the fluid laterally around the tank
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© Informa UK Ltd, 2006
Module 2 – GROWTH OPERATIONS
A Practical Guide to Biopharmaceutical Manufacturing
The drive for the impeller system is supplied by an external motor and can be attached to either the
top of the vessel or the bottom. In both cases, the design of the impeller shaft seals is important
because they must prevent the entry of any contaminants and in the case of a bottom driven impeller
arrangement, the leakage of the broth itself. Sealing methods often use condensate seals fed by clean
process steam to ensure sterile operation.
Organisms that are easily damaged must be grown in vessels using impellers and baffles that do not
generate shear forces able destroy them. The total power consumption of a large-scale stirred tank
bioreactor is typically in the range of 1–4W/L of working volume.
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2.2
Stirred tank bioreactors use mechanical devices e.g. impellers
to agitate the growth medium
Attachment dependent
Some mammalian cell lines require a surface to grow successfully and therefore cannot be grown in
suspension. Consequently, the chosen bioreactor must provide sufficient surface area for the
organisms to grow on whilst allowing access to both oxygen and nutrients and removal of waste.
Attachment dependent culture of mammalian cells is a subject of continued research, and a number of
different methods have been developed to provide a stable environment with a large surface area that
also enables good heat and mass transfer. However, attachment dependent growth is often restricted
to small-scale operation due to complex environmental controls and handling difficulties not present in
suspension bioreactors. The technique, though, is widely used in the production of viruses that require
a mammalian cell base and small production volumes.
Common examples of cell culture equipment include:
•
Roller bottles
•
Plates – including T-flasks, cell factories, and cell cubes
•
Hollow fibre
•
Suspended micro-carriers
•
Packed beds
Once grown, cells can be harvested by mechanical or chemical methods. Mechanical methods, such as
scraping, are best suited to relatively simple systems such as roller bottles. More complex systems
require the use of a chemical or biological agent to break the bond between the cells and the support.
Two common agents are EDTA and trypsinase. EDTA is a chemical that works by removing the calcium
and magnesium used by cells to form bonds with the support. Trypsinase is a digestive enzyme that
cleaves proteins to break the binding. In both cases, the chemical agent can be removed using
diafiltration. However trypsination can cause damage to many proteins and may not be suitable if the
product is secreted.
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In attachment dependent cultures, the chosen bioreactor must
provide sufficient surface area for the organisms to grow on
whilst allowing access to the necessary nutrients and the
removal of waste
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Module 2 – GROWTH OPERATIONS
2.2.1
A Practical Guide to Biopharmaceutical Manufacturing
Roller bottles
Roller bottles are cylindrical containers, partly filled with growth medium, in which attachment
dependent cultures can be grown on the interior surface. The bottles are slowly rotated on mechanical
rollers or racks so that the organisms bound to the surface alternately come into contact with the gas
space and the growth media. The scale of the operation is determined by the number of bottles used,
because scaling of the individual roller bottles does not give rise to the required surface area to
volume. Each roller bottle is typically 1–2L in volume and is usually filled with media to approximately
25% of the total volume.
The bottles are inoculated whilst rotating at a low rate, e.g., 0.1–0.3 revolutions/minute, to allow
attachment to take place. Once the culture has successfully anchored to the bottle surface, the rolling
rate is increased to approximately 1–2 revolutions/minute. During the growth phase, the medium may
be exchanged on a regular basis. Yields are highly dependent on the specific application and organism,
with typical results of approximately 5x105 cells/mL of bottle volume. The relative surface area of roller
bottles may be increased by ‘pleating’ the interior surface; however this can make the harvesting of
cells more complex. Typical surface area to volume ratio for roller bottles is 0.02–0.07 m2/L.
The greatest disadvantage of roller bottles systems is the high labour requirement for the complex and
repetitive handling tasks associated with each bottle. However, this limitation has been somewhat
alleviated by the development of automated robotic handling.
2.2.2
Plates
Plate-based bioreactors consist of flat surfaces used to culture cells. A number of different systems of
varying complexity exist with an emphasis on disposable applications. The simplest forms of platebased cell culture use flat dishes or T-flasks (Figure 2.3). However, the surface area to volume ratio of
these methods is low, and they are not suited to product manufacture.
Figure 2.3: T-flasks
Stacks of plates can be used to provide a greater surface area to volume ratio and allow more efficient
production of cells within a given volume. An example are Nalgene cell factories (Figure 2.4), available
with up to 40 plates offering a maximum suggested working volume of 8L. Stacked plates are often
used to grow the inocula for larger production bioreactors.
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© Informa UK Ltd, 2006
Module 2 – GROWTH OPERATIONS
A Practical Guide to Biopharmaceutical Manufacturing
A more-complex variant, the Cell Cube, manufactured by Corning Life Sciences, uses cubic modules of
stacked plates. These modules are then placed in specially designed containers and aerated medium is
perfused through the container and the cubes within. The modular design allows easy scale-up, and
stacked plate systems typically give surface area to volume ratios of approximately 0.17m2/L or higher,
depending on the configuration.
Figure 2.4: Nalgene cell factories
In addition to adding more plates, the surface area to volume ratio may also be increased by using
coiled sheets. Spacers can be used to create space between the coils for cell growth; the surface area
to volume ratio is approximately 0.4m2/L.
The cultured cell mass is harvested through washing with a solution such as Tween, a non-ionic
detergent that does not denature the cells.
2.2.3
Hollow fibres
Hollow fibre culture uses beds of synthetic capillary fibres to which the cells may attach (Figure 2.5).
Cells are introduced to the spaces between the fibres where they can grow. Aerated medium is then
pumped through the bed of fibres. Pores in the fibres allow the medium to supply the cells with oxygen
and nutrients. The bundles of fibres give a large surface area upon which the cells may attach and
allow growth at high cell concentrations. Typical surface area to volume ratios for hollow fibre
bioreactors are approximately 3 m2/L.
Figure 2.5: A schematic of a hollow fibre bioreactor
Product/
Cells Out
Media
Feed
Media
Outlet
Hollow
Fibre
These high cell densities can result in different conditions along the length of the reactor, and
gradients in key nutrients, oxygen, etc., can result. These variations may lead to a loss in productivity
or product quality.
The use of hollow fibre beds is not restricted to attachment dependent cell lines. Suspension cells may
also be grown in the space between the fibres. The properties of the fibres can be chosen to control
product harvest. Pores can be sized to allow passage of the product through the fibre wall and into the
main medium flow, although the product is more commonly retained on the cell side of the fibre wall
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