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.
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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
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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 108per 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 IPT 36 2010 10/3/11 14:29 Page 62
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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 IPT 36 2010 10/3/11 14:30 Page 63
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.
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Timo Keijzerjoined 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: [email protected]
Erik Kakesis 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 Halsemais 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.
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