Pharmaceutical

future science group

Pharm. Bioprocess. (2013) 1(2), 167–177

16710.4155/PBP.13.19 © 2013 Future Science Ltd ISSN 2048-9145

Review

Single-use bioreactors are commonly used in the biopharmaceutical industry today, however, they are mostly limited to mammalian cell culture processes. For microbial processes, concepts including the CELL-tainer® technology provide comparable oxygen mass transfer such as in stirred tank reactors. Data obtained with 15 and 120  l working volumes indicate excellent performance with Escherichia  coli and Corynebacterium glutamicum cultures. Therefore, this type of single-use bioreactor is applicable in biopharmaceutical processes, and also in a seed train for bulk chemicals production such as amino acid production. It is expected that single-use technologies will be applied ever more frequently in microbial-fed batch cultivation processes in combination with improved monitoring and control.

Single-use bioreactors (SUBs, also often re-ferred to as disposable bioreactors) are nowa-days widely applied in the biopharmaceutical industry. The scale is not restricted to labo-ratory use, as reactors with working volumes up to the m³-scale exist and can also be used for good manufactur practice processes. SUBs offer unique advantages when compared with traditional glass or stainless steel bioreactors. As the SUBs are presterilized (by gamma ir-radiation), a complex infrastructure (such as autoclaves or clean-in-place/steam-in-place installations) is no longer needed. This saves investment and space and reduces operational costs, but moreover, shortens the timelines for validation significantly, thus making it possi-ble to introduce the final products faster into the market. With the increasing number of therapeutic candidates (monoclonal antibod-ies, other biotherapeutic proteins, diagnostics, human and veterinary vaccines) entering the market, a flexible production environment is needed.

In the last decade, the application of dispos-able technologies has increased considerably in the biopharmaceutical industry. In 1987, Kybal described a single-use type of bioreactor

for cultivation of plant cells [1]. A step towards innovation was achieved by the release of the first single-use wave-mixed bioreactor for the cultivation of shear-sensitive cells by Singh in 1999 [2]. The wide spread of the technology has led to numerous optimiz ations and dif-ferent approaches of SUBs for alternative ap-plications in science and industry.

Due to the ongoing development in cell line engineering, the cultivation becomes more robust and more efficient, which de-mands a decreasing cultivation volume. In recent years, titers of cell cultures in industri-ally relevant bioprocesses have increased from 0.05 to over 10 g/l [3]. Hence, bioreactors of smaller capacity are of increasing interest. This stimulates the demand for, and imple-mentation of, SUBs, which usually possess limits in size compared with steel vessels. SUBs are also becoming available on larger scales. For selected products with low market requirements, production applying single-use equipment is becoming possible even at com-mercial scales, which adds much more flex-ibility to a production plant.

For a long time, the application of SUBs was restricted to mammalian cell culture

Single-use bioreactors for microbial cultivation

Nico MG Oosterhuis*1, Peter Neubauer2 & Stefan Junne2

1CELLution Biotech BV, Dr AF Philipsweg 15A, 9403AC Assen, The Netherlands 2Chair of Bioprocess Engineering, Institute of Biotechnologie, Technische Universität Berlin, Germany *Author for correspondence: E-mail: nico.oosterhuis@cellutionbiotech.com

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168 future science groupPharm. Bioprocess. (2013) 1(2)

processes. The reason lies in the restriction of the gas–liquid oxy-gen mass transfer (kLa), which of-ten could not compete with any traditional stirred tank bioreactor. However, approximately 40–45% of all biopharmaceutical products that are in Phase III of clinical de-velopment are based on microbial processes (Boehringer Ingelheim [unpublished data, 2009]); this concerns a wide variety of products, such as therapeutic and diagnostic proteins. In biopharmaceutical processes, for the production of small molecular weight therapeutic proteins, human

and veterinary vaccines and numerous other prod-ucts, microbial-based processes are applied. There-fore, there is a strong demand for single-use equip-ment that is applicable to microbial processes, as the advantages of using SUBs also account for these kinds of processes.

Moreover, single-use equipment can be applied in fermentation processes for antibiotics, amino acids and enzymes production to reduce the risk of contamina-tion in seed-trains. In these types of products, a failure of 2–5% of the production batches due to contamina-tion is not unusual, which is mainly caused by con-taminations already present during the seeding stage. Another suitable application of disposables is in the field of marine process development, where the high chloride content causes corrosion of typical stainless

steel. Coatings or resistant steel brands are expensive and costs might not be affordable at early stages of product and process development.

This review will provide an overview of SUBs for microbial cultivation processes above a liquid volume of 1 l, and of current developments broadening the future application of these types of bioreactors in this specific field.

Different types of SUBsSUBs are made of disposable parts, usually bags and sensors, connections for tubing (pH-regulating agents, feed and others) and gas filters. The disposable parts are delivered presterilized to the customer. The steril-ization is usually performed using gamma irradiation at intensities between 25 and 50 kGy. Hence, vali-dation of cleaning and sterilization is not any longer the responsibility of the customer who uses the bio-reactor, but the supplier who provides the disposable presterilized material.

Never the less, there are disadvantages of SUBs, which are usually a low gas–liquid oxygen mass trans-fer, prolonged mixing times and, in general, a poor understanding of fluid flow in comparison with tradi-tional steel-stirred tank reactors.

A low gas–liquid oxygen mass transfer is critical for fungal, yeast or bacterial processes (which require oxy-gen transfer levels of at least 150 mmol/l/h and which might comprise of viscous media). Up to now, scale-up of many systems to pilot scale has been achieved up to a volume of 2000 l (Table 1) [4]. This volume is al-ready sufficient to cover the annual demand of several

Table 1. Overview of commercially available disposable bioreactors.Reactor type Working

volume (l)Type of bag Type of mixing Supplier (location) kLa

(h-1)Ref.

Wave Bioreactor™ 1–200 Pillow Rocking GE Healthcare Biosciences (PA, USA) <10 [101]

BIOSTAT® RM 1–100 Pillow Rocking Sartorius Stedim Biotech (Göttingen, Germany)

<10 [102]

Appliflex 1–25 Pillow Rocking Applikon Biotechnology (Schiedam, The Netherlands)

<40 [103]

CELL-tainer® – 20 0.2–25 Pillow or square 2D rocking CELLution Biotech (Assen, The Netherlands) >400 [104]

CELL-tainer – 200 5–200 Pillow or square 2D rocking CELLution Biotech >400 [104]

Cultibag BIOSTAT STR200

50–200 Tankliner Stirred Sartorius Stedim Biotech >150 [102]

Single use bioreactor 50–1000 Tankliner Stirred Thermo-Fischer (Hyclone) (UT, USA) <40 [105]

XDR single-use bioreactor

40–2000 Tankliner Stirred GE Healthcare/Xcellerex (MA, USA) <20 [106]

Mobius® CellReady 50–200 Tankliner Stirred Merck Millipore (MA, USA) <70 [107]

Nucleo single-use bioreactor

50–100 Square 3D Paddle ATMI/Pierre Guerin (Hoegaarden, Belgium) <20 [108]

SBX reactor/ BIOSTAT ORB

200 Tankliner Orbital Shaker Kuhner/Excell Gene (Birsfelden/Monthey, both Switzerland)/Sartorius Stedim Biotech

– [102,109]

CellMaker Regular 1–50 Bubble column Rotating sparger

Cellexus (Cambridgeshire, UK) <10 [110]

Key Terms

Single-use bioreactor: Uses a bag made of plastic and is disposed of after use. Mainly applied for mammalian cell processes in the biopharmaceutical industry.

Gas–liquid oxygen mass transfer coefficient (kLa): Liquid side mass transfer coefficient (kL) multiplied by the specific gas–liquid interfacial area (a). Also expressed as oxygen transfer coefficient. The parameter determining the gas–liquid transfer capacity of the bioreactor.

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Single-use bioreactors for microbial cultivation

mammalian-based production processes. Most of the systems are not only equipped with a disposable bag, but also with sensors for monitoring typical process parameters such as flow rate, temperature, pH and dissolved oxygen (DO), similar to stirred tanks [5]. However, many of the sensors are disposable, which demands sensitivity and stability losses due to their specialized construction and size.

Systems that are mostly applied are the wave-mixed (‘rocking’) type bioreactors (WAVE [GE Healthcare Life Sciences; PA, USA], BIOSTAT® CultiBag RM [Sartorius; Göttingen, Germany], Ap-pliFlex [ Applikon Biotech; Schiedam, The Nether-lands], and XRS 20 [PALL Life Sciences; NY, USA]), shaken and stirred SUBs (Thermo Scientific Hyclone Single-Use Bioreactor S.U.B [Thermo-Fisher; UT, USA], BIOSTAT CultiBag STR [Sartorius], XDR™ [GE/XCellerex®; MA, USA], New Brunswick Cel-liGEN® BLU [Eppendorf; Hamburg, Germany], and DASbox® [DASGIP®; Jülich, Germany]). An over-view of commercially available systems was recently presented in [5].

In wave-mixed SUBs, the bag is mounted on a rock-ing platform that moves vertically around a fixed cen-tral point up to angles of maximal 15°. Another type of rocking bioreactor suitable for microbial cultivation is the CELL-tainer® concept by CELLution Biotech (Assen, The Netherlands; exclusively distributed in the EU and northern American markets by Charter Medical Inc. [NC, USA]). In contrast to the WAVE bioreactor, the bag platform follows a 2D vertical and horizontal movement. This leads to an obviously high-er power input, since the mass transfer coefficients in these systems are much higher than at ‘classical’ rock-

ing platforms (Table 1). Therefore, this bioreactor is also suitable for microbial applications.

During the last 5 years, several other types of SUBs with characteristic power input have also been intro-duced, for example the Mobius® CellReady (Millipore) bioreactor in which agitation is provided by a pitch-blade impeller on a levitating bearing-less drive in combination with a membrane-based micro-sparger; the IntegrityTM PadReactorTM (ATMI; Hoegaarden, Belgium), which is a square bag equipped with a pad-dle in which a gas sparger is integrated in the paddle; an orbital shaking bioreactor (SBX orbital shaker [Kuhner; Birsfelden Switzerland]; BIOSTAT® ORB [Sartorius]) where the whole cylindrical bag is mov-ing on a shaking-platform; and the Air-Wheel® air-driven mixing system [PBS Biotech® ]. Table 1 provides a nonextensive overview of commercially available SUBs and their features with the focus on gas–liquid oxygen mass transfer capacities.

All these SUBs have been designed primarily for mam-malian cell culture applications. As a mammalian cell culture, with a viable cell density of 20 × 106 cells ml-1,

Figure 1. CELL-tainer® single-use bioreactor. Photos courtesy of CELLution Biotech.

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Figure 2. Gas–liquid oxygen mass transfer in the CELL-tainer® bioreactor up to 15 l scale and up to 150 l scale. Reproduced with permission from [6].

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170 future science groupPharm. Bioprocess. (2013) 1(2)

only consumes approximately 3–4 mmol/l/h of oxygen, a gas–liquid oxygen mass transfer coefficient (K

La) for

oxygen of kLa >15 h-1 is sufficient for most applications.

In contrast, for microbial cultures, the required gas–liquid oxygen mass transfer coefficient for oxygen has to be much higher. In these cultures, an oxygen uptake rate of at least 150 mmol/l/h is common and necessary to compete with traditional stirred tank reactors – it is the basis for reaching a reliable cell density. For such a volumetric oxygen consumption, the gas–liquid oxygen mass transfer coefficient has to be at least k

La = 400 h-1.

Due to the 2D rocking motion in combination with a pillow-like design of the culture bag, the CELL-tainer

SUB (Figure 1) creates a closed loop of the bag movement. Experiments for the investigation of the gas–liquid oxy-gen mass transfer (dynamic method in water at 20°C), show values of over 400 h-1 at a 5–15 l scale. These values are also achievable with the scaled-up version at 100–150 l (Figure 2) [6].

In the CELL-tainer (at a working volume of 10 l) running at 20 rpm, a power input of P/V = 360 W/m3 is achieved, resulting in a k

La = 100 h-1 [CELLution Bio-

tech, Unpublished Data]. The Wave type SUB has a power input of P/V = 50 W/m3 [7]. Considering the van’t Riet [8] equation for the relation between power input and k

La, it can be assumed that the gas–liquid oxygen mass

transfer in the CELL-tainer is more efficient due to the 2D rocking motion. Since k

La is a function of (P/V)0,7,

according to the van’t Riet equation [8] an increase of the power input by a factor of approximately 7 should result in an increase of the k

La value by a factor 4.

However, in practice, an increase compared with the traditional wave-type bioreactor by a factor of 10 has been observed instead.

This is one reason for the accelerated kLa values that

are achieved in comparison with other wave-mixed sys-tems. Another reason for this might be a prolonged film formation and, hence, larger surface area between the

Figure 3. Eddy movement in a CELL-tainer® bioreactor with 120 l filling volume (simulation model) from the right to the left edge. The present rocking direction of the tray is indicated by the black arrows.

Current expansion process (shake flasks in incubators, wave bioreactors)

Vial thaw Culture initiation

1:4

Seed step 1

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37°C/2.5 min 250 ml SF 500 ml SF 2 × 1 l SF1:4

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Vial thaw Culture initiation

250 ml SF

40 ml

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CELL-tainer® (160 ml–25 l)

1:10

Bioreactor

Seed step 4

1 × 25/50 l

Figure 4. Typical seed train for a mammalian cell culture process.SF: Shake flask. Reproduced with permission from [9].

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Single-use bioreactors for microbial cultivation

liquid and gas phase due to the horizontal movement. The distinct increase of the k

La value in the larger sys-

tem is possibly caused by additional eddy formation. This eddy formation starting from the edges of the bag reaches up to the middle of the bag before collapsing (Figure 3). It is appearing at a filling level that is not too low (early collapse of the eddy) and not too large (no eddy formation observable). The eddy formation likely increases the power input. However, detailed studies of the fluid dynamics have to be performed. Since the k

La

values at 12 and at 120 l are comparable at both scales, the growth performance should be as well.

The bag is situated in a closed incubator, thus en-suring very accurate temperature control, which is cru-cial for the application at microbial cultures. Due to the higher volumetric consumption rates, the control of the temperature is much more demanding than at cell line proliferation. For efficient cooling, which is a prerequisite for systems suitable for microbial cultures, the CELL-tainer is supplied with a cooling plate in the bottom of the moving tray.

Applications for working volumes above 100 lThe rocking based SUBs – in mammalian cell culture – are not only applied in laboratories for screening and pro-duction of nonclinical material, but also in seed trains. A step-wise scale-up in rocking systems is performed up to working volumes of 200–500 l. A standard seed pro-cess includes at least six steps before sufficient amounts of cells are available to inoculate the production reac-tor (Figure 4). Investigations performed by Shire HGT (MA, USA) showed that at least three seed steps can be reduced when applying the CELL-tainer SUB, in which so-called ‘expansion blocks’ are used [9]. This offers the possibility to create a variable working volume in one and the same bag from 0.15–25 l. In this way, handling and risk of contamination is significantly reduced.

Since mammalian cell culture processes are becom-ing more productive due to advances in cell line engi-neering and process development, cell culture titers in fed-batch processes have increased from 0.05 to over 10 g/l during the last 15 years [3]. Even productivities of above 25 g/l are reported at the production of a mono-clonal antibody using the XD®-process with the human PER.C6®-cells [10]. As a result, scale-up is restricted to volumes of 2000 l, since there is no market demand in the biopharmaceutical industry, which still dictates reactor development.

Bags are always manufactured with material of USP class VI. They are exposed to a qualification process concerning extractables and leachables, as well as steril-ity and integrity. This information is usually available from the vendors for established systems and from the suppliers of the developed film materials. This whole

qualification procedure leads to technical limits in the volume size that is applicable. However, SUB design for the application of microbial cultivation is already avail-able on the market. These systems have to be equipped with suitable monitoring and control in order to be able to compete with traditional stirred tank systems.

At scale-up, mixing times of SUBs are usually af-fected, since a concomitant increase of power input is restricted for mechanical reasons. For example, mixing times in the 120 l CELL-tainer are approximately a fac-tor of five larger than in the 12 l scale [6]. However, as long as the characteristic time for pH-control is in bal-ance with the mixing performance, no process-relevant gradients of pH might be expected. For temperature control (cooling) no gradients are expected either.

Monitoring SUBsSince one approach to overcome the mentioned disad-vantages of SUBs for application to microbial processes is a suitable strategy for appropriate feeding to control growth (nutrient-limiting fed-batch), monitoring be-comes even more important. Monitoring capabilities of SUBs are crucial, since sensors connected to these sys-tems have to withstand gamma-irradiation and must be comparably cheap due to their disposable application. In the main, sensors are applied in two ways: systems that are cheap and an integral part of the bioreactor and, thus, disposable; and conventional sensors that are identical to the ones applied in stirred tank reac-tors made of steel. The requirements of the first group led to the development of sensors that are often not as accurate as traditionally applied sensor systems in steel stirred tanks. For single-use, dissolved-oxygen and pH sensors based on optical principles are applied, which are noninvasive. A spot is printed on the foil, on to which reacting material is bound. Most principles rely on phase fluorometry. Light (typically at a wavelength of 480 nm) is conducted to a spot of fluorescent dye at the inner side of the bag wall via optical fiber technol-ogy. The corresponding excited fluorescence (usually a wavelength of 520 nm) depends on the DO and pH and is collected through the optical fiber towards a photo-sensor [11]. Thus, the sensor unit is decoupled from the parts to be sterilized; only the dye is exposed to gamma-irradiation. Sensor systems for the measurement of dis-solved carbon dioxide are currently in use, also relying on an optical determination method (PreSens [Regens-burg, Germany] and Polestar Technologies [MA, USA] among others).

One drawback of this method is the loss of sensitiv-ity at the spot due to photobleaching, which restricts the lifetime. The change in intensity measurement of the fluorescent signal due to gamma irradiation leads to a complex calibration procedure. The second group

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generally contains any electrode that can be coupled to a traditional stirred tank system. One example is the PadReactor from ATMI where these electrodes are con-nected via aseptic KleenpakTM connectors from PALL. The sensors are reusable and rely on amperometric DO and potentiometric pH measurement. Since they are not directly in contact with the culture media, presteril-ization is not necessary, which simplifies the calibration procedures. This approach has the advantage of being fully compatible to monitoring in steel stirred tank re-actors with respect to accuracy and life time of sensors. The CELL-tainer system uses electrochemical pH de-tection and a micro-amperometric DO measurement. While the pH electrode is disposable, the DO sensor can be reused many times, as only the sensor membrane makes up part of the disposable bag. The sensors are mounted in small cups at the bottom of the bag, which offers the advantage that they are covered with liquid at all times, even at low filling volumes and under angled conditions. Since neither electrode relys on fluorescent measurements, they are also characterized by longer life times.

Beside standard parameters, sensors for impedance measurements are also applied, with which the capaci-tance of cells is determined. This provides information on the cell vitality and can be related to cell growth.

Sensors are available for SUBs from Fogale Nanotech and Aber Instruments. The devices can be coupled with flow-through cells and the electrodes can also be attached to the bag surface.

Noninvasive radio frequency measurements have been proven to yield a spectrum, which can be coupled to the estimation of temperature and conductivity [12]. By applying multivariate data ana lysis, these data are gained from one single sensor system. The impact of environmental conditions on measurements is reduced, proving the suitability of the approach in long-term measurements. Nacke et al. describe the application of a microwave spectroscopy sensor suitable for continu-ous monitoring of the fermentation media in SUBs. Between frequencies of 0.3–10 GHz, the permittivity and conductivity is determined non invasively by the in-tegration of a dielectric window as mechanical port [13].

The determination of process-relevant metabolites, such as glucose, glutamate and lactate, is performed with biochemical sensors, which comprise of immobi-lized enzymes and are distributed gamma-radiated (e.g. C-CIT sensors that are available for single-use systems). While these sensors can be connected directly to the liquid phase, several other microfluidic systems have also been developed that are mounted outside the reac-tor and are generally applicable for SUBs. These micro-fluidic devices are hosting the immobilized biochemi-cal sensors. Moser et al. describe array systems for the simultaneous determination of glucose, lactate, gluta-mine and glutamate. Flow rates were in the range of 0.1 to 100 µl/min. Since these flow-through micro devices are suitable for gamma-irradiation, integration in dis-posable concepts is possible [14]. Other methods com-mon for the online detection of metabolites are near-IR and Fourier-transformed IR spectroscopy. These methods are similar in that they are not all suitable for a direct combination with single-use systems due to their complexity and expensive equipment. However, these sensors are coupled to bypass pipes (coupled to common cell-retention systems at SUBs) to measure the cell-free media or, in the case of near-IR, directly connected when suitable barriers such as translucent membranes are installed. Coupling sensors to SUBs is described by Furey from a practical point of view [15].

The current state-of-the-art and the steady develop-ment of the sensor technique for disposable systems is broadening the possibilities for improved monitoring and control, shortening the gap to traditional stirred tank bioreactors and enabling the application for microbial cultivation.

Microbial application in wave-mixed SUBsAs has been stated above, microbial cultures require more oxygen and more intensive mixing than mam-

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Figure 5. Comparison of Escherichia coli growth in a classical stirred fermenter and the single-use CELL-tainer® bioreactor. Reproduced with permission from [22].

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Single-use bioreactors for microbial cultivation

malian cell culture processes, despite the application of fed-batch procedures. In wave-mixed bioreactors with working volumes of 100 l, k

La values of k

La < 10 h-1 and

mixing times > 100 s-1 are reported [7]. Typical power consumptions of up to 0.05 W/l for a 2 l wave-mixed bioreactor have been examined. Usually bioreactors for microbial applications are equipped with stirrers transferring 2 W/l and more [8].

When applying a sparger into the liquid (10 l work-ing volume in a 20 l wave-bag), some improvement can be achieved. In this way, a gas–liquid oxygen mass transfer coefficient of k

La = 60 h-1 has been reported. A

Wave SUB was applied in this way for the cultivation of Saccharomyces cerevisiae [16]. In a 5 l batch culture, oxygen limitation was not observed until a biomass concentration of 8 g/l, due to blending of the inlet gas with oxygen. At increased volumes, the oxygen limita-tion was severe and could not be avoided when baffles were applied additionally.

Several other applications of bacterial cultivation in wave-mixed bioreactors are reported in the literature. In a batch procedure, a final biomass concentration below 2 g/l was achieved in a BIOSTAT CultiBag RM system when cultivating Escherichia coli before oxygen deple-tion [17]. In a nutrient-limited E. coli fed-batch approach an optical density (OD) of OD

600 = 30 (10 g/l of dry

biomass) was achieved. The applied recombinant E. coli RB791 strain expressed a heterologous alcohol dehy-drogenase. Hence, recombinant protein expression was performed successfully in the SUB. At these conditions, the feed was controlled with the enzymatic substrate re-lease method EnBase® (BioSilta; Oulu, Finland) [18], while no external feeding was applied. This approach is very suitable when monitoring systems are not reliable enough. This methodology can compensate for some drawbacks existing with current monitoring systems at disposable bioreactors.

The application of SUBs for the growth of Coryne-bacterium diphtheriae for vaccine production was inves-tigated by Ullah et al. [19]. In the BIOSTAT CultiBag RM reactor, a final cell density of OD

590 = 5 (which can

be considered as approximately 2 g/l of dry biomass) compared with OD

590 = 7.3 in an aerated stirred tank

bioreactor was measured [19]. Hitchcock described the cultivation of a recombinant Listeria monocytogenes for vaccine production. The product was used for Phase II clinical trials. A BIOSTAT CultiBag RM bioreactor was applied due to easier validation in comparison to a traditional steel bioreactor. When the filling volume was reduced to 5 l, a sufficient gas–liquid oxygen mass transfer at a final OD

600 of 12 was achieved [20].

A suitable further application of wave-mixed SUBs is the utilization for inoculum cultivations. This applica-tion is beneficial, while a smaller cell density is usually

targeted than in production processes. A study in a WAVE SUB with 5 l cultivation volume described the growth of an E. coli culture towards the final targeted OD

600 of 15. In this range of the cell density, the oxygen

transfer was sufficient to avoid oxygen limitation [21]. Due to the larger k

La values at the CELL-tainer in

comparison with other wave-mixed bioreactors, it is especially useful for the application for microbial cul-tivation. Several investigations have been performed to prove the performance of the CELL-tainer in the cultivation of E. coli. In a study performed by Sanofi-Pasteur, it has been demonstrated that at a working volume of 7 l, both growth and protein production performance in the CELL-tainer are comparable with the data obtained in a traditional stirred tank reactor, as depicted in Figure 5 [22]. In batch cultures, densi-

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Figure 6. Growth of Corynebacterium glutamicum in the CELL-tainer® compared with a stirred fermenter. CT: Cell-tainer; STR: Stirred fermenter. Courtesy CELLution biotech.

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Figure 7. Growth of Escherichia coli. Growth of Escherichia coli in a pH 7.0-controlled, nutrient-limiting, fed-batch cultivation in 12 l (closed symbols and straight line) and 120 l (open symbols and dashed line) in the CELL-tainer® single-use bioreactor. The cell dry weight is indicated as circles, the glucose concentration as triangles. More information available from [6].

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174 future science groupPharm. Bioprocess. (2013) 1(2)

ties of OD600

= 60 were obtained and in the fed-batch mode even OD

600 = 90 was achieved. This study proves

the potential of the application of SUBs for microbial cultures.

The CELL-tainer SUB is applied in a seed train for production of lysine by Corynebacterium glutamicum. Usually, during first steps in such a large-scale pro-cess, shake flasks are applied. As the working volume of a shake flask is limited to approximately 1 l, several flasks have to be pooled to inoculate one large fermenter (>10 m3). Both pooling of the different flasks and flask-to-flask variability may lead to problems in such a seed train. Compared with shake flasks, a bioreactor reduces handling and provides a controlled preculture (pH and DO control). The seed process can also be performed in a standard stirred (autoclavable or in situ sterilizable) fermenter. However, for this setup, a more complex infrastructure is needed, fermenters demand cleaning (manual operation) and there is less flexibility. In com-parison with a traditional stirred tank reactor, growth in the CELL-tainer shows comparable results (Figure 6).

As discussed in the introduction, the application of SUBs for marine cultures has benefits in comparison with the cultivation in steel stirred tank reactors. Re-cently, own studies of the CELL-tainer technology for the cultivation of heterotrophic microalgae in marine media has been proven to be sucessful. Although nu-trient-limitation cannot be processed in this case, the gas transfer was sufficient to achieve a cell dry weight of over 45 g/l at maximum growth rates of 0.05 h-1. Lehm-an et al. performed a study in which the suitability of SUBs for application with phototrophic algae was in-vestigated [23]. The different systems that were observed were a BIOSTAT CultiBag RM, which was equipped with red and white LEDs, the wave-mixed AppliFlex (Applikon Biotechnology) with white light LEDs, and the orbital shaken CultiBag RM on the Multitron Cell shaker (Infors HT; Bottmingen, Switzerland) with white fluorescent tubes. In all systems, similar cell den-sities were achieved, such as in stirred, helical tubular and an airlift photobioreactor.

Another field of application of SUBs for microbial cultivation is in anaerobic processes. The bacterium Eubacterium ramulus was used as model organism in a culture of a working volume of 10 l in a BIOSTAT CultiBag RM SUB [24]. In this culture, a final wet bio-mass concentration of 60–70 g/l was achieved, which is comparable with results obtained in steel stirred tank reactors.

Scale-up of microbial cultivations in the CELL-tainer bioreactor Recently, an investigation showed the potential of growing E. coli cultures in a larger cultivation volume

of 120 l [6]. In this study, a genetically transformed E. coli B21-Gold strain (Agilent Technologies; DE, USA) was used that encoded a maltogenic amylase. The cultivation was running in a nutrient-limiting fed-batch mode. At a working volume of 12 l and only after 32 h, a final OD

600 of over 130 was reached. At the tenfold

scale a similar biomass yield was obtained, proving the successful scale-up of the process (Figure 7).

Microbial application in stirred tank SUBs & orbital shakersDue to the beneficial height-to-diameter ratio and less required ground space, bags with larger volumes than 500 l are available for stirred SUBs. Usually, these re-actors are equipped with several blade impellers and microspargers such as the XDR from XCellerex (GE Life Sciences) and the BIOSTAT CultiBag STR from Sartorius. Galliher et al. report a 50 l cultivation of E. coli in a XDR-50 SUB [25,26]. In this process, a cell density of OD

600 = 120 (dry cell weight of 40 g/l) was

achieved. In addition, Pseudomonas fluorescens was cultivated in the XDR. In this case, a dry cell weight of over 100 g/l was obtained. In this study, the process performance was described as similar to cultivations in conventional nondisposable stirred tank reactors [24,25]. When a sufficient control strategy is applied, similar yields as those obtained in stainless stirred tank reactors can be achievable in microbial cultiva-tions using SUBs. Based on the maximum oxygen transfer capacity available in the BIOSTAT CultiBag RM and BIOSTAT Cultibag STR, the medium feed is adapted to a linear feed instead of an exponential feed for a wave-mixed BIOSTAT CultiBag RM and an exponential feed for a stirred BIOSTAT CultiBag STR was applied. An OD of up to OD

600 = 140 was

achievable at E. coli cultivations [27].The orbitally shaken bioreactor SBX from Kuhner

has no inlet parts due to the fact that the whole reactor is shaken as a consistent scale-up from culture flasks [28]. Although k

La values are approximately 10 h-1,

which is sufficient for achieving satisfying results with mammalian cell cultures [29], our own studies have also yielded good results at cultures with higher oxygen demand, such as heterotrophic microalgae. Another study reports the successful cultivation of transgenic tobacco cells. After 6 days of cultivation, a dry cell weight of 12 g/l was achieved at a volume of 10 l [30].

The characterization of these reactors, with respect to the power input and gas–liquid oxygen mass trans-fer, requires the consideration of further constraints than for directly stirred SUBs. This has been ac-complished at the laboratory scale [31] and is ongoing for larger orbital shakers, when, for example, liquid-

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Single-use bioreactors for microbial cultivation

film formation and the concomitant increase in the surface-to-volume ratio has to be considered.

ConclusionThe advantages in applying SUBs for microbial pro-cesses are equal to those for mammalian processes. As microbial processes usually running for 1–5 days, time for cleaning and sterilizing of the bioreactor can exceed 20% of the process time. Hence, besides complex in-frastructure, more fermentation capacity is also needed when applying steel bioreactors compared with single-use systems. For laboratory-scale fermentations, usually autoclavable glass fermenters are applied. Although the infrastructure is less demanding, time needed to clean and to autoclave such glass fermenters is laborious. A yearly cost saving of €50,000 can easily be achieved when using a single-use fermenter (10 l working vol-ume, compared with an autoclavable fermenter) [32].

To date, presently available SUBs are less suitable for cultivation of microbial cultures. Due to limitation in mass transfer and mixing, cell densities are limited in those SUBs that are designed for mammalian cell culture. A comparison of different techniques is not easy to perform, since only the gas transfer and mixing times can be compared applying standard techniques. Further considerations such as the power input are not trivial to perform and demand time. Some work has al-ready been performed for the BIOSTAT CultiBag RM wave-mixed reactor [5]. However, for other systems such as the SBX or the CELL-tainer, research is still ongoing.

The CELL-tainer SUB is available for working vol-umes up to 150 l. Based on growth data of E. coli and C. glutamicum cultures, it can be concluded that this reac-tor type is very suitable for microbial cultivation. Due to a 2D rocking motion, the turbulence in the liquid is intensified and dissipated power per volume is com-parable with standard stirred fermenters for micro bial

processes. This results in a significantly higher (factor 10–20) K

La. The combination of intelligent software

sensor control strategies and currently improving (dis-posable) sensors will lead to a reduction of drawbacks. For example, the correct settings for nutrient-limiting fed-batch procedures can be achieved by appropriate sensor-based control. Limitations of sensor application such as correct biomass determination can be circum-vented by these strategies, when affordable and – even more important – reliable and robust sensors for dispos-able bioreactors are widely applicable.

Future perspectiveThe benefits of using SUBs in biopharmaceutical pro-cessing are significant. Costs of production can be re-duced, and due to a greatly simplified infrastructure, industry can shorten the timelines of getting products into the clinical trial phase, which is a key driving factor.

The availability of SUBs for microbial cultivations widens their potential, not only in biopharmaceutical processing, but also as a preculture bioreactor for bulk fermentations. It is expected that in the next 5–10 years, the bioreactor landscape will be covered with many more single-use systems.

DisclaimerThe views expressed in the article are the author’s own and do not necessarily reflect the views of Future Science Ltd.

Financial & competing interests disclosureNMG Oosterhuis is CTO/CSO of CELLution Biotech BV, being the company who developed the CELL-tainer®. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the sub-ject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary

Background » Single-use bioreactors (SUBs) are applied mainly in mammalian cell culture processes. » New developments widen the application of SUBs to microbial processes, and even bulk industrial

fermentations.Different types of SUBs » Since their introduction, many different types of SUBs have appeared. » Most commonly used are the ‘rocking-type’ SUBs as well as the ‘stirred type’.

Monitoring of SUBs » Proper process control in SUBs is a key element in a good manufacturing practice environment. » Various methods have been, and are, applied, with the main complicating factor being the need for the

sensors to be resistent to gamma irradiation.SUBs for microbial application » Gas–liquid oxygen mass transfer should be sufficient to support biomass formation in SUBs comparable with

traditional stainless steel or glass (stirred) bioreactors. » The CELL-tainer® SUB shows kLa values that support microbial fermentations and has been shown to be

scaleable.

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176 future science groupPharm. Bioprocess. (2013) 1(2)

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