Advances in the Design of Bioreactor Systems

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Bioprocessing

By Timo Keijzer and Developments in areas such as miniaturisation, data collection software Erik Kakes at Applikon and sensor/actuator equipment are changing the way bioreactor systems Biotechnology BV, and Emo van Halsema at are designed. Halotec Instruments BV Bioreactors are closed systems in which a biological processes for biopharmaceuticals are based on the stirred process can be carried out under controlled, tank bioreactor. The scale-up process from laboratory- to environmental conditions. A bioreactor system production-sized systems is therefore based on this comprises a bioreactor, sensors and actuators, a control design as well. This cylindrical bioreactor uses a top- or system and software to monitor and control the bottom-mounted rotating mixing system. Generally, the conditions inside the bioreactor. tank has an aspect ratio of between 1:1.5 (for mammalian cell culture) and 1:3 (for microbial Designing a bioreactor system involves mechanical, fermentations). Baffles can be installed to enhance electrical and bioprocess engineering. Since standard mixing where the baffle diameter is typically one tenth of bioreactors can be used in a variety of applications, the the tank diameter. The impeller can either be a marine design process should be organised in such a way that impeller for axial mixing of the cell culture – having a systems can be used under the strictest of regulations. diameter of between one-third and one-half of the tank These design rules are described in the cGMP and GAMP diameter – or multiple Rushton turbines for gas bubble guidelines, as well as the American Society of Mechanical breaking and axial mixing in microbial cultures. Gas is Engineers (ASME) BioProcessing Equipment (BPE) typically introduced below the mixing impeller, and guidelines for the design of bioprocess equipment. liquid additions are done from the top of the bioreactor. Stirred tank bioreactors are available from 0.05 litres up Typical applications of bioreactors can be found in the to 100 cubic metres in volume. production of pharmaceuticals, food bio-based materials (such as poly-lactic acid), bio-fuels – and also in Other bioreactor designs include the following: waste treatment. Photo Bioreactors TYPES OF BIOREACTOR A photo bioreactor incorporates a light source to provide photonic energy input into the reactor. They are The stirred tank bioreactor is the classical design of generally used for the cultivation of photosynthesising Figure 1: Autoclavable stirred tank bioreactor bioreactor and is still the most widely used. Most organisms (plants, algae and bacteria). Industrial-scale system production facilities and FDA-approved production 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.

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

Figure 2: Mini bioreactors with 250 ml total volume

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Figure 3: Single-use cGMP photo bioreactor

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

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gel-filled miniature pH sensors and polarographic more vigorous way of mixing is needed. A miniature oxygen sensors with an outer diameter of only 6 mm. direct drive was developed for this purpose; the drive can These sensors enable the reliable measurement of pH run continuously at 2,000 rpm to guarantee proper and and dissolved oxygen over a longer period (weeks or scalable mixing and mass transfer on a miniature scale. months). The pH sensor can be used from pH2 up to pH12, making it applicable to a wider range of processes DESK-BASED PROCESS DESIGN than other miniature sensors (such as fluorophors) that cannot measure below pH5 or above pH8. The massive amount of data generated with these small- scale instruments needs to be interpreted, and so must be On the actuator side, the challenge is to add small visualised in order for all the information to be digested. amounts of liquids under controlled conditions; this is Data needs to be gathered using smart data collection particularly important when working with continuous software; such software can compare data across different additions of media. Adding a droplet of concentrated cultivation platforms and guide the user to select the medium at the three-litre scale does not influence the optimal settings for specific strains. Data mining and culture, but one droplet at a 50 ml volume makes a other techniques enable the analysis of large amounts of significant difference in nutrient concentration. A special data, as well as the identification of correlations and sterilisable injection valve was developed to add nano- underlying structures. litre droplets of liquid to the culture on a continuous basis; this enables the smooth addition of (highly Mathematical models that describe cell growth as a concentrated) liquids into the bioreactor. function of medium composition allow the user to design cultivation media by computer. This approach Most miniature, stirred tank bioreactors rely on a provides an insight into the effects of changing specific magnetic stirrer bar for agitation. This is acceptable for medium conditions (such as the buffer capacity) on cell mammalian cell cultivation where the mixing and growth and product formation. In addition, the effects of oxygen demands are limited, but for microbial cultures a 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 Timo Keijzer joined Applikon Biotechnology BV (Schiedam, running experiments from the desk, and mobile access to Netherlands) in 2001 as a Product Manager. In 1999, he obtained experiments; this mobile access allows the user to interact a degree from the Department of BioProcess Engineering at with the processes at any time and from any location. Wageningen University and Research Center (Netherlands). In 2000, he continued his studies at the Boku University (Vienna, Mobile access is available through smart phones or a Austria) in the Department of Applied Microbiology, specialising in tablet PC; of course, the access is limited to authorised ultrasonic cell separation (using sound waves to separate cells users through a strict security policy. from culture medium for perfusion). Email: [email protected] Based on these new technologies, the development time for Erik Kakes is International Sales & Marketing Director at Applikon Biotechnology BV. He joined Applikon in 1988 as a Project new pharmaceuticals can be greatly reduced, resulting in Manager and then moved into sales via the R&D department. lower R&D costs. Smaller bioreactors can even reduce the In 2008, he acquired the ownership of Applikon with Arthur bench space needed for experiments – ultimately resulting Oudshoorn and Jaap Oostra through a management buy-out. in the possibility of smaller laboratories and reducing the Erik graduated in 1984 from the Van’t Hoff Institute (Rotterdam, investment needed for expensive laboratory space. Netherlands), with a specialisation in Biochemistry. From 1984 to 1988, he worked for the sugar-producing company Cosun optimising xanthan gum production. CONCLUSION

Emo van Halsema is Managing Director of Halotec Instruments Over recent decades, changes in bioreactor system design BV (Veenendaal, Netherlands) – a company that he set up and have focused mainly on the software and control side of which specialises in the development of innovative, fully-automated measuring systems and process equipment for the life sciences these systems, while more recently the single-use industry. He has developed a software suite for bringing together revolution has changed the design of pilot plants and process data from multiple sources, incorporating modeling, production bioreactors for cell culture. A new area for process control and data storage. His team of collaborating change is the miniaturisation of the bioreactor system, engineers work on developing solutions to any biotech/engineering/software and new technologies are now available for sensors and technical challenge – from data gathering to data analysis. Emo graduated from the actuators. With more data being generated in a shorter Delft University of Technology (Netherlands) where he stayed for 10 years before setting up Halotec. time period, the time to market for new drugs will be greatly reduced.

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