Bioprocessing

processes for biopharmaceuticals are based on the stirredtank bioreactor. The scale-up process from laboratory- toproduction-sized systems is therefore based on thisdesign as well. This cylindrical bioreactor uses a top- orbottom-mounted rotating mixing system. Generally, thetank has an aspect ratio of between 1:1.5 (formammalian cell culture) and 1:3 (for microbialfermentations). Baffles can be installed to enhancemixing where the baffle diameter is typically one tenth ofthe tank diameter. The impeller can either be a marineimpeller for axial mixing of the cell culture – having adiameter of between one-third and one-half of the tankdiameter – or multiple Rushton turbines for gas bubblebreaking and axial mixing in microbial cultures. Gas istypically introduced below the mixing impeller, andliquid additions are done from the top of the bioreactor.Stirred tank bioreactors are available from 0.05 litres upto 100 cubic metres in volume.

Other bioreactor designs include the following:

Photo Bioreactors A photo bioreactor incorporates a light source to providephotonic energy input into the reactor. They aregenerally used for the cultivation of photosynthesisingorganisms (plants, algae and bacteria). Industrial-scalephoto 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 aregrown on moist, solid particles. The spaces between theparticles contain a continuous gas phase and a minimalamount of water. The majority of solid-statefermentation (SSF) processes involve filamentous fungi,although some also involve bacteria or yeasts. Solid-statefermentation is mainly used in food processes.

Bubble Column Bioreactors These are tall column bioreactors where gas is introducedinto the bottom section for mixing and aerationpurposes.

Developments in areas such as miniaturisation, data collection softwareand sensor/actuator equipment are changing the way bioreactor systemsare designed.

60 Innovations in Pharmaceutical Technology

Advances in the Design of Bioreactor Systems

Bioreactors are closed systems in which a biologicalprocess can be carried out under controlled,environmental conditions. A bioreactor systemcomprises a bioreactor, sensors and actuators, a controlsystem and software to monitor and control theconditions inside the bioreactor.

Designing a bioreactor system involves mechanical,electrical and bioprocess engineering. Since standardbioreactors can be used in a variety of applications, thedesign process should be organised in such a way thatsystems can be used under the strictest of regulations.These design rules are described in the cGMP and GAMPguidelines, as well as the American Society of MechanicalEngineers (ASME) BioProcessing Equipment (BPE)guidelines for the design of bioprocess equipment.

Typical applications of bioreactors can be found in theproduction 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 ofbioreactor and is still the most widely used. Mostproduction facilities and FDA-approved production

By Timo Keijzer and Erik Kakes at ApplikonBiotechnology BV, andEmo van Halsema atHalotec Instruments BV

Figure 1: Autoclavablestirred tank bioreactorsystem

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Air-Lift Bioreactors Similar to bubble column reactors, these differ by thefact that they contain a draft tube. There are two types ofdraft tube: an inner tube (air-lift bioreactor with aninternal loop); or an external tube (air-lift bioreactorwith an external loop). The draft tube improvescirculation and oxygen transfer, and equalises shearforces in the reactor. Airlift bioreactors are available fromlaboratory scale up to full production scale.

Hollow Fibre Cartridges Hollow fibres are small tube-like filters sealed into acartridge shell so that cell culture medium pumpedthrough the end of the cartridge will flow through theinside of the fibre, while the cells are grown on theoutside of the fibre. Hollow fibres provide atremendous amount of surface area in a small volume.Cells grow on and around the fibres at densities ofgreater than 1 x 108 per ml. Hollow fibre cell culture isthe only means to culture cells at in vivo-like celldensities. Cell culture at high densities can achieve a 10to 100 times higher concentration of secreted productcompared with classic batch processes. The scalabilityof the hollow fibre system is limited, however, and sothese types of bioreactor are mainly used at thelaboratory scale.

Rocking Bag Bioreactors Approximately 15 years ago, the rocking bag bioreactorwas introduced as the first single-use bioreactor. Thissystem relies on the rocking motion of the bioreactorholder 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 coolingcapacity of such systems.

Stem Cell Bioreactors A recent development is the stem cell bioreactor.Numerous designs exist for these types of bioreactorbut the goal is the same – to cultivate and differentiatestem cells. There are no commercial products on themarket yet, but several joint research programmesbetween 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 ofsuccessful designs.

TRENDS

Currently, several trends can be identified with regard tobioreactor design. Of course, recent years have beendominated by new developments in single-use bioreactortechnology. This has focused mainly on small and largerproduction volume bioreactors (50 litres and upwards),and aims to reduce the initial investment costs of newproduction facilities. Another trend focuses on the R&Dside of biotechnology, which is also cost-driven; thisincludes the scale down of bioreactors to millilitre andeven microlitre volumes – the ultimate goal being toreduce the time to market for new drugs. This approachfocuses on obtaining more data at an earlier stage ofprocess development, and enables more efficientdecisions to be made during the process of selectingspecific strains or media for further process developmentor production. This set-up requires a large number ofcultures running in parallel under identical, controlledconditions. In the next stage of scale-up, processdevelopment also needs to be optimised and made astime-efficient as possible. Again, this means that a largenumber of cultures need to be run in parallel under

Figure 2: Mini bioreactors with 250 ml total volume

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different conditions to select the optimal growth andproduction conditions for the selected strains.

This work was classically carried out in three-litrebioreactors on the laboratory bench; the reasoningbehind this was that the results found in the bench-topsystem would be scalable to pilot plant and productionlevel. The three-litre scale was the smallest volume thatwould still allow an equal mixing regime, and enable useof the same sensor and actuator technology as those atthe larger scales.

MINIATURISATION

Recent developments in sensor and actuator technologyhave enabled the further scale down of bioreactors, whilestill maintaining the required scalability to pilot andproduction volumes.

The German company PreSensGmbH has developed fluorophor-based sensor technology for thenon-invasive measurement of pHand dissolved oxygen. Thistechnology has been successfully applied in microtiterplates, turning these devices into well-controlledcultivation systems. Cultivation volumes are in themillilitre range, and mixing is achieved by placing themicrotiter plates on a shaker. This is a good first step in thedevelopment of small bioreactors, but a control system(liquid additions and so on) has yet to be developed.

At Applikon Biotechnology, we recently introduced abioreactor for scalable operation to volumes as low as 50ml, with miniaturised classical sensor and actuatortechnology. A number of breakthrough technologieswere developed to realise this; these included sterilisable

Figure 3: Single-use cGMP photo bioreactor

Figure 4: Stirred tank photo bioreactor

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gel-filled miniature pH sensors and polarographicoxygen sensors with an outer diameter of only 6 mm.These sensors enable the reliable measurement of pHand dissolved oxygen over a longer period (weeks ormonths). The pH sensor can be used from pH2 up topH12, making it applicable to a wider range of processesthan other miniature sensors (such as fluorophors) thatcannot measure below pH5 or above pH8.

On the actuator side, the challenge is to add smallamounts of liquids under controlled conditions; this isparticularly important when working with continuousadditions of media. Adding a droplet of concentratedmedium at the three-litre scale does not influence theculture, but one droplet at a 50 ml volume makes asignificant difference in nutrient concentration. A specialsterilisable injection valve was developed to add nano-litre droplets of liquid to the culture on a continuousbasis; this enables the smooth addition of (highlyconcentrated) liquids into the bioreactor.

Most miniature, stirred tank bioreactors rely on amagnetic stirrer bar for agitation. This is acceptable formammalian cell cultivation where the mixing andoxygen demands are limited, but for microbial cultures a

more vigorous way of mixing is needed. A miniaturedirect drive was developed for this purpose; the drive canrun continuously at 2,000 rpm to guarantee proper andscalable 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 bevisualised in order for all the information to be digested.Data needs to be gathered using smart data collectionsoftware; such software can compare data across differentcultivation platforms and guide the user to select theoptimal settings for specific strains. Data mining andother techniques enable the analysis of large amounts ofdata, as well as the identification of correlations andunderlying structures.

Mathematical models that describe cell growth as afunction of medium composition allow the user todesign cultivation media by computer. This approachprovides an insight into the effects of changing specificmedium conditions (such as the buffer capacity) on cellgrowth and product formation. In addition, the effects ofby-products can be examined before any laboratorytesting is done. Other time-saving features of modernsoftware are remote access to view and analyse actualrunning experiments from the desk, and mobile access toexperiments; this mobile access allows the user to interactwith the processes at any time and from any location.Mobile access is available through smart phones or atablet PC; of course, the access is limited to authorisedusers through a strict security policy.

Based on these new technologies, the development time fornew pharmaceuticals can be greatly reduced, resulting inlower R&D costs. Smaller bioreactors can even reduce thebench space needed for experiments – ultimately resultingin the possibility of smaller laboratories and reducing theinvestment needed for expensive laboratory space.

CONCLUSION

Over recent decades, changes in bioreactor system designhave focused mainly on the software and control side ofthese systems, while more recently the single-userevolution has changed the design of pilot plants andproduction bioreactors for cell culture. A new area forchange is the miniaturisation of the bioreactor system,and new technologies are now available for sensors andactuators. With more data being generated in a shortertime period, the time to market for new drugs will begreatly reduced.

64 Innovations in Pharmaceutical Technology

Timo Keijzer joined Applikon Biotechnology BV (Schiedam,Netherlands) in 2001 as a Product Manager. In 1999, he obtaineda degree from the Department of BioProcess Engineering atWageningen University and Research Center (Netherlands). In2000, he continued his studies at the Boku University (Vienna,Austria) in the Department of Applied Microbiology, specialising inultrasonic 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 ApplikonBiotechnology BV. He joined Applikon in 1988 as a ProjectManager and then moved into sales via the R&D department. In 2008, he acquired the ownership of Applikon with ArthurOudshoorn 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 InstrumentsBV (Veenendaal, Netherlands) – a company that he set up andwhich specialises in the development of innovative, fully-automatedmeasuring systems and process equipment for the life sciencesindustry. He has developed a software suite for bringing togetherprocess data from multiple sources, incorporating modeling,process control and data storage. His team of collaborating

engineers work on developing solutions to any biotech/engineering/softwaretechnical challenge – from data gathering to data analysis. Emo graduated from theDelft University of Technology (Netherlands) where he stayed for 10 years beforesetting up Halotec.

PreSens Anzeige SFR GIT Laboratory Journal _03.indd 1 07.02.2011 16:32:45

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