The stirred tank bioreactor is the most widely used bioreactor design. Developments in areas like miniaturization are changing bioreactor design. Miniature bioreactors under development can operate at volumes as low as 50ml while maintaining scalability and using miniaturized sensor and actuator technologies. These developments may reduce the time and costs required for new drug development.

1. Bioprocessing
processes for biopharmaceuticals are based on the stirred
tank bioreactor. The scale-up process from laboratory- to
production-sized systems is therefore based on this
design as well. This cylindrical bioreactor uses a top- or
bottom-mounted rotating mixing system. Generally, the
tank has an aspect ratio of between 1:1.5 (for
mammalian cell culture) and 1:3 (for microbial
fermentations). Baffles can be installed to enhance
mixing where the baffle diameter is typically one tenth of
the tank diameter. The impeller can either be a marine
impeller for axial mixing of the cell culture – having a
diameter of between one-third and one-half of the tank
diameter – or multiple Rushton turbines for gas bubble
breaking and axial mixing in microbial cultures. Gas is
typically introduced below the mixing impeller, and
liquid additions are done from the top of the bioreactor.
Stirred tank bioreactors are available from 0.05 litres up
to 100 cubic metres in volume.
Other bioreactor designs include the following:
Photo Bioreactors
A photo bioreactor incorporates a light source to provide
photonic energy input into the reactor. They are
generally used for the cultivation of photosynthesising
organisms (plants, algae and bacteria). Industrial-scale
photo bioreactors can also be open pond systems;
obviously, these cannot be considered as closed systems,
and so are more sensitive to environmental influences.
Solid-State Bioreactors
These are used for processes where microorganisms are
grown on moist, solid particles. The spaces between the
particles contain a continuous gas phase and a minimal
amount of water. The majority of solid-state
fermentation (SSF) processes involve filamentous fungi,
although some also involve bacteria or yeasts. Solid-state
fermentation is mainly used in food processes.
Bubble Column Bioreactors
These are tall column bioreactors where gas is introduced
into the bottom section for mixing and aeration
purposes.
Developments in areas such as miniaturisation, data collection software
and sensor/actuator equipment are changing the way bioreactor systems
are designed.
60 Innovations in Pharmaceutical Technology
Advances in the Design
of Bioreactor Systems
Bioreactors are closed systems in which a biological
process can be carried out under controlled,
environmental conditions. A bioreactor system
comprises a bioreactor, sensors and actuators, a control
system and software to monitor and control the
conditions inside the bioreactor.
Designing a bioreactor system involves mechanical,
electrical and bioprocess engineering. Since standard
bioreactors can be used in a variety of applications, the
design process should be organised in such a way that
systems can be used under the strictest of regulations.
These design rules are described in the cGMP and GAMP
guidelines, as well as the American Society of Mechanical
Engineers (ASME) BioProcessing Equipment (BPE)
guidelines for the design of bioprocess equipment.
Typical applications of bioreactors can be found in the
production of pharmaceuticals, food bio-based materials
(such as poly-lactic acid), bio-fuels – and also in
waste treatment.
TYPES OF BIOREACTOR
The stirred tank bioreactor is the classical design of
bioreactor and is still the most widely used. Most
production facilities and FDA-approved production
By Timo Keijzer and
Erik Kakes at Applikon
Biotechnology BV, and
Emo van Halsema at
Halotec Instruments BV
Figure 1: Autoclavable
stirred tank bioreactor
system

2. 62 Innovations in Pharmaceutical Technology
Air-Lift Bioreactors
Similar to bubble column reactors, these differ by the
fact that they contain a draft tube. There are two types of
draft tube: an inner tube (air-lift bioreactor with an
internal loop); or an external tube (air-lift bioreactor
with an external loop). The draft tube improves
circulation and oxygen transfer, and equalises shear
forces in the reactor. Airlift bioreactors are available from
laboratory scale up to full production scale.
Hollow Fibre Cartridges
Hollow fibres are small tube-like filters sealed into a
cartridge shell so that cell culture medium pumped
through the end of the cartridge will flow through the
inside of the fibre, while the cells are grown on the
outside of the fibre. Hollow fibres provide a
tremendous amount of surface area in a small volume.
Cells grow on and around the fibres at densities of
greater than 1 x 108
per ml. Hollow fibre cell culture is
the only means to culture cells at in vivo-like cell
densities. Cell culture at high densities can achieve a 10
to 100 times higher concentration of secreted product
compared with classic batch processes. The scalability
of the hollow fibre system is limited, however, and so
these types of bioreactor are mainly used at the
laboratory scale.
Rocking Bag Bioreactors
Approximately 15 years ago, the rocking bag bioreactor
was introduced as the first single-use bioreactor. This
system relies on the rocking motion of the bioreactor
holder to mix a liquid volume contained in a plastic bag.
This type of bioreactor is mainly used for cell cultivation,
due to the low oxygen transfer rates and limited cooling
capacity of such systems.
Stem Cell Bioreactors
A recent development is the stem cell bioreactor.
Numerous designs exist for these types of bioreactor
but the goal is the same – to cultivate and differentiate
stem cells. There are no commercial products on the
market yet, but several joint research programmes
between industry and universities are focusing on
the development of stem cell bioreactor systems.
Applikon Biotechnology has participated in several
of these projects and has developed a number of
successful designs.
TRENDS
Currently, several trends can be identified with regard to
bioreactor design. Of course, recent years have been
dominated by new developments in single-use bioreactor
technology. This has focused mainly on small and larger
production volume bioreactors (50 litres and upwards),
and aims to reduce the initial investment costs of new
production facilities. Another trend focuses on the R&D
side of biotechnology, which is also cost-driven; this
includes the scale down of bioreactors to millilitre and
even microlitre volumes – the ultimate goal being to
reduce the time to market for new drugs. This approach
focuses on obtaining more data at an earlier stage of
process development, and enables more efficient
decisions to be made during the process of selecting
specific strains or media for further process development
or production. This set-up requires a large number of
cultures running in parallel under identical, controlled
conditions. In the next stage of scale-up, process
development also needs to be optimised and made as
time-efficient as possible. Again, this means that a large
number of cultures need to be run in parallel under
Figure 2: Mini bioreactors
with 250 ml total volume

3. 63
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different conditions to select the optimal growth and
production conditions for the selected strains.
This work was classically carried out in three-litre
bioreactors on the laboratory bench; the reasoning
behind this was that the results found in the bench-top
system would be scalable to pilot plant and production
level. The three-litre scale was the smallest volume that
would still allow an equal mixing regime, and enable use
of the same sensor and actuator technology as those at
the larger scales.
MINIATURISATION
Recent developments in sensor and actuator technology
have enabled the further scale down of bioreactors, while
still maintaining the required scalability to pilot and
production volumes.
The German company PreSens
GmbH has developed fluorophor-
based sensor technology for the
non-invasive measurement of pH
and dissolved oxygen. This
technology has been successfully applied in microtiter
plates, turning these devices into well-controlled
cultivation systems. Cultivation volumes are in the
millilitre range, and mixing is achieved by placing the
microtiter plates on a shaker. This is a good first step in the
development of small bioreactors, but a control system
(liquid additions and so on) has yet to be developed.
At Applikon Biotechnology, we recently introduced a
bioreactor for scalable operation to volumes as low as 50
ml, with miniaturised classical sensor and actuator
technology. A number of breakthrough technologies
were developed to realise this; these included sterilisable
Figure 3: Single-use cGMP photo bioreactor
Figure 4: Stirred
tank photo bioreactor

4. gel-filled miniature pH sensors and polarographic
oxygen sensors with an outer diameter of only 6 mm.
These sensors enable the reliable measurement of pH
and dissolved oxygen over a longer period (weeks or
months). The pH sensor can be used from pH2 up to
pH12, making it applicable to a wider range of processes
than other miniature sensors (such as fluorophors) that
cannot measure below pH5 or above pH8.
On the actuator side, the challenge is to add small
amounts of liquids under controlled conditions; this is
particularly important when working with continuous
additions of media. Adding a droplet of concentrated
medium at the three-litre scale does not influence the
culture, but one droplet at a 50 ml volume makes a
significant difference in nutrient concentration. A special
sterilisable injection valve was developed to add nano-
litre droplets of liquid to the culture on a continuous
basis; this enables the smooth addition of (highly
concentrated) liquids into the bioreactor.
Most miniature, stirred tank bioreactors rely on a
magnetic stirrer bar for agitation. This is acceptable for
mammalian cell cultivation where the mixing and
oxygen demands are limited, but for microbial cultures a
more vigorous way of mixing is needed. A miniature
direct drive was developed for this purpose; the drive can
run continuously at 2,000 rpm to guarantee proper and
scalable mixing and mass transfer on a miniature scale.
DESK-BASED PROCESS DESIGN
The massive amount of data generated with these small-
scale instruments needs to be interpreted, and so must be
visualised in order for all the information to be digested.
Data needs to be gathered using smart data collection
software; such software can compare data across different
cultivation platforms and guide the user to select the
optimal settings for specific strains. Data mining and
other techniques enable the analysis of large amounts of
data, as well as the identification of correlations and
underlying structures.
Mathematical models that describe cell growth as a
function of medium composition allow the user to
design cultivation media by computer. This approach
provides an insight into the effects of changing specific
medium conditions (such as the buffer capacity) on cell
growth and product formation. In addition, the effects of
by-products can be examined before any laboratory
testing is done. Other time-saving features of modern
software are remote access to view and analyse actual
running experiments from the desk, and mobile access to
experiments; this mobile access allows the user to interact
with the processes at any time and from any location.
Mobile access is available through smart phones or a
tablet PC; of course, the access is limited to authorised
users through a strict security policy.
Based on these new technologies, the development time for
new pharmaceuticals can be greatly reduced, resulting in
lower R&D costs. Smaller bioreactors can even reduce the
bench space needed for experiments – ultimately resulting
in the possibility of smaller laboratories and reducing the
investment needed for expensive laboratory space.
CONCLUSION
Over recent decades, changes in bioreactor system design
have focused mainly on the software and control side of
these systems, while more recently the single-use
revolution has changed the design of pilot plants and
production bioreactors for cell culture. A new area for
change is the miniaturisation of the bioreactor system,
and new technologies are now available for sensors and
actuators. With more data being generated in a shorter
time period, the time to market for new drugs will be
greatly reduced.
64 Innovations in Pharmaceutical Technology
Timo Keijzer joined Applikon Biotechnology BV (Schiedam,
Netherlands) in 2001 as a Product Manager. In 1999, he obtained
a degree from the Department of BioProcess Engineering at
Wageningen University and Research Center (Netherlands). In
2000, he continued his studies at the Boku University (Vienna,
Austria) in the Department of Applied Microbiology, specialising in
ultrasonic cell separation (using sound waves to separate cells
from culture medium for perfusion). Email: info@applikon-biotechnology.com
Erik Kakes is International Sales & Marketing Director at Applikon
Biotechnology BV. He joined Applikon in 1988 as a Project
Manager and then moved into sales via the R&D department.
In 2008, he acquired the ownership of Applikon with Arthur
Oudshoorn and Jaap Oostra through a management buy-out.
Erik graduated in 1984 from the Van’t Hoff Institute (Rotterdam,
Netherlands), with a specialisation in Biochemistry. From 1984
to 1988, he worked for the sugar-producing company Cosun optimising
xanthan gum production.
Emo van Halsema is Managing Director of Halotec Instruments
BV (Veenendaal, Netherlands) – a company that he set up and
which specialises in the development of innovative, fully-automated
measuring systems and process equipment for the life sciences
industry. He has developed a software suite for bringing together
process data from multiple sources, incorporating modeling,
process control and data storage. His team of collaborating
engineers work on developing solutions to any biotech/engineering/software
technical challenge – from data gathering to data analysis. Emo graduated from the
Delft University of Technology (Netherlands) where he stayed for 10 years before
setting up Halotec.