Pharmaceutical Review

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

Single-use bioreactors for microbial cultivation

Single-use bioreactors are commonly used in the biopharmaceutical industry today, Nico MG Oosterhuis*1, Peter however, they are mostly limited to mammalian cell culture processes. For microbial Neubauer2 & Stefan Junne2 processes, concepts including the CELL-tainer® technology provide comparable 1CELLution Biotech BV, Dr AF oxygen mass transfer such as in stirred tank reactors. Data obtained with 15 and Philipsweg 15A, 9403AC Assen, The Netherlands 120 l working volumes indicate excellent performance with Escherichia coli and 2Chair of Bioprocess Engineering, Corynebacterium glutamicum cultures. Therefore, this type of single-use bioreactor is Institute of Biotechnologie, Technische applicable in biopharmaceutical processes, and also in a seed train for bulk chemicals Universität Berlin, Germany production such as amino acid production. It is expected that single-use technologies *Author for correspondence: will be applied ever more frequently in microbial-fed batch cultivation processes in E-mail: nico.oosterhuis@ cellutionbiotech.com combination with improved monitoring and control.

Single-use bioreactors (SUBs, also often re- for cultivation of plant cells [1] . A step towards ferred to as disposable bioreactors) are nowa- innovation was achieved by the release of the days widely applied in the biopharmaceutical first single-use wave-mixed bioreactor for the industry. The scale is not restricted to labo- cultivation of shear-sensitive cells by Singh in ratory use, as reactors with working volumes 1999 [2]. The wide spread of the technology up to the m³-scale exist and can also be used has led to numerous optimiz­ations and dif- for good manufactur practice processes. SUBs ferent approaches of SUBs for alternative ap- offer unique advantages when compared with plications in science and industry. traditional glass or stainless steel bioreactors. Due to the ongoing development in cell As the SUBs are presterilized (by gamma ir- line engineering, the cultivation becomes radiation), a complex infrastructure (such as more robust and more efficient, which de- autoclaves or clean-in-place/steam-in-place mands a decreasing cultivation volume. In installations) is no longer needed. This saves recent years, titers of cell cultures in industri- investment and space and reduces operational ally relevant bioprocesses have increased from costs, but moreover, shortens the timelines for 0.05 to over 10 g/l [3]. Hence, bioreactors of validation significantly, thus making it possi- smaller capacity are of increasing interest. ble to introduce the final products faster into This stimulates the demand for, and imple- the market. With the increasing number of mentation of, SUBs, which usually possess therapeutic candidates (monoclonal antibod- limits in size compared with steel vessels. ies, other biotherapeutic proteins, diagnostics, SUBs are also becoming available on larger human and veterinary vaccines) entering the scales. For selected products with low market market, a flexible production environment is requirements, production applying single-use needed. equipment is becoming possible even at com- In the last decade, the application of dispos- mercial scales, which adds much more flex- able technologies has increased considerably ibility to a production plant. in the biopharmaceutical industry. In 1987, For a long time, the application of SUBs Kybal described a single-use type of bioreactor was restricted to mammalian cell culture

future science group 10.4155/PBP.13.19 © 2013 Future Science Ltd ISSN 2048-9145 167 Review Oosterhuis, Neubauer & Junne

Key Terms processes. The reason lies in the steel. Coatings or resistant steel brands are expensive Single-use bioreactor: Uses restriction of the gas–liquid oxy- and costs might not be affordable at early stages of

a bag made of plastic and gen mass transfer (kLa), which of- product and process development. is disposed of after use. ten could not compete with any This review will provide an overview of SUBs for Mainly applied for mammalian traditional stirred tank bioreactor. microbial cultivation processes above a liquid volume cell processes in the biopharmaceutical industry. However, approximately 40–45% of 1 l, and of current developments broadening the Gas–liquid oxygen mass of all biopharmaceutical products future application of these types of bioreactors in this that are in Phase III of clinical de- specific field. transfer coefficient (kLa): Liquid side mass transfer coefficient velopment are based on microbial (k ) multiplied by the specific L processes (Boehringer Ingelheim Different types of SUBs gas–liquid interfacial area (a). Also expressed as oxygen [unpublished data, 2009]); this concerns SUBs are made of disposable parts, usually bags and transfer coefficient. The a wide variety of products, such as sensors, connections for tubing (pH-regulating agents, parameter determining the gas– therapeutic and diagnostic proteins. feed and others) and gas filters. The disposable parts liquid transfer capacity of the In biopharmaceutical processes, for are delivered presterilized to the customer. The steril- bioreactor. the production of small molecular ization is usually performed using gamma irradiation weight therapeutic proteins, human at intensities between 25 and 50 kGy. Hence, vali- and veterinary vaccines and numerous other prod- dation of cleaning and sterilization is not any longer ucts, microbial-based processes are applied. There- the responsibility of the customer who uses the bio- fore, there is a strong demand for single-use equip- reactor, but the supplier who provides the disposable ment that is applicable to microbial processes, as the presterilized material. advantages of using SUBs also account for these kinds Never the less, there are disadvantages of SUBs, of processes. which are usually a low gas–liquid oxygen mass trans- Moreover, single-use equipment can be applied in fer, prolonged mixing times and, in general, a poor fermentation processes for antibiotics, amino acids and understanding of fluid flow in comparison with tradi- enzymes production to reduce the risk of contamina- tional steel-stirred tank reactors. tion in seed-trains. In these types of products, a failure A low gas–liquid oxygen mass transfer is critical for of 2–5% of the production batches due to contamina- fungal, yeast or bacterial processes (which require oxy- tion is not unusual, which is mainly caused by con- gen transfer levels of at least 150 mmol/l/h and which taminations already present during the seeding stage. might comprise of viscous media). Up to now, scale-up Another suitable application of disposables is in the of many systems to pilot scale has been achieved up field of marine process development, where the high to a volume of 2000 l (Table 1) [4]. This volume is al- chloride content causes corrosion of typical stainless ready sufficient to cover the annual demand of several

Table 1. Overview of commercially available disposable bioreactors.

Reactor type Working Type of bag Type of mixing Supplier (location) kLa Ref. volume (l) (h-1) Wave Bioreactor™ 1–200 Pillow Rocking GE Healthcare Biosciences (PA, USA) <10 [101] BIOSTAT® RM 1–100 Pillow Rocking Sartorius Stedim Biotech (Göttingen, <10 [102] Germany) Appliflex 1–25 Pillow Rocking Applikon Biotechnology (Schiedam, <40 [103] The Netherlands) 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 50–200 Tankliner Stirred Sartorius Stedim Biotech >150 [102] STR200 Single use bioreactor 50–1000 Tankliner Stirred Thermo-Fischer (Hyclone) (UT, USA) <40 [105] XDR single-use 40–2000 Tankliner Stirred GE Healthcare/Xcellerex (MA, USA) <20 [106] bioreactor Mobius® CellReady 50–200 Tankliner Stirred Merck Millipore (MA, USA) <70 [107] Nucleo single-use 50–100 Square 3D Paddle ATMI/Pierre Guerin (Hoegaarden, Belgium) <20 [108] bioreactor SBX reactor/ 200 Tankliner Orbital Shaker Kuhner/Excell Gene (Birsfelden/Monthey, both – [102,109] BIOSTAT ORB Switzerland)/Sartorius Stedim Biotech CellMaker Regular 1–50 Bubble column Rotating Cellexus (Cambridgeshire, UK) <10 [110] sparger

168 Pharm. Bioprocess. (2013) 1(2) future science group Single-use bioreactors for microbial cultivation Review 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® Figure 1. CELL-tainer single-use bioreactor. Photos courtesy of CELLution Biotech. CultiBag RM [Sartorius; Göttingen, Germany], Ap- pliFlex [ Applikon Biotech; Schiedam, The Nether- ing platforms (Table 1). Therefore, this bioreactor is also lands], and XRS 20 [PALL Life Sciences; NY, USA]), suitable for microbial applications. shaken and stirred SUBs (Thermo Scientific Hyclone During the last 5 years, several other types of SUBs Single-Use Bioreactor S.U.B [Thermo-Fisher; UT, with characteristic power input have also been intro- USA], BIOSTAT CultiBag STR [Sartorius], XDR™ duced, for example the Mobius® CellReady (Millipore) [GE/XCellerex®; MA, USA], New Brunswick Cel- bioreactor in which agitation is provided by a pitch- liGEN® BLU [Eppendorf; Hamburg, Germany], and blade impeller on a levitating bearing-less drive in DASbox® [DASGIP®; Jülich, Germany]). An over- combination with a membrane-based micro-sparger; view of commercially available systems was recently the IntegrityTM PadReactorTM (ATMI; Hoegaarden, presented in [5]. Belgium), which is a square bag equipped with a pad- In wave-mixed SUBs, the bag is mounted on a rock- dle in which a gas sparger is integrated in the paddle; ing platform that moves vertically around a fixed cen- an orbital shaking bioreactor (SBX orbital shaker tral point up to angles of maximal 15°. Another type [Kuhner; Birsfelden Switzerland]; BIOSTAT® ORB of rocking bioreactor suitable for microbial cultivation [Sartorius]) where the whole cylindrical bag is mov- is the CELL-tainer® concept by CELLution Biotech ing on a shaking-platform; and the Air-Wheel® air- (Assen, The Netherlands; exclusively distributed in driven mixing system [PBS Biotech®].­ Table 1 provides the EU and northern American markets by Charter a nonextensive overview of commercially available Medical Inc. [NC, USA]). In contrast to the WAVE SUBs and their features with the focus on gas–liquid bioreactor, the bag platform follows a 2D vertical and oxygen mass transfer capacities. horizontal movement. This leads to an obviously high- All these SUBs have been designed primarily for mam- er power input, since the mass transfer coefficients in malian cell culture applications. As a mammalian cell these systems are much higher than at ‘classical’ rock- culture, with a viable cell density of 20 × 106 cells ml-1,

600 600

500 500 -1

-1 400 400

)

a (h a )

a (h a 300 L

L 300

k k 200 200 35 30 100 30 100 25 20 0 25 0 6 80 m rpm 90 15 8 20 100 rp Liquid volum10 e (l) Liqui 110 10 12 d volume120 (l)130 14 15 140 150

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].

future science group www.future-science.com 169 Review Oosterhuis, Neubauer & Junne

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 -1 is achieved, resulting in a k a = 100 h [CELLution Bio- Figure 3. Eddy movement in a CELL-tainer® bioreactor with 120 l filling L volume (simulation model) from the right to the left edge. The present tech, Unpublished Data]. The Wave type SUB has a power 3 rocking direction of the tray is indicated by the black arrows. input of P/V = 50 W/m [7]. Considering the van’t Riet [8] equation for the relation between power input and

only consumes approximately 3–4 mmol/l/h of oxygen, kLa, it can be assumed that the gas–liquid oxygen mass

a gas–liquid oxygen mass transfer coefficient (KLa) for transfer in the CELL-tainer is more efficient due to the -1 0,7 oxygen of kLa >15 h is sufficient for most applications. 2D rocking motion. Since kLa is a function of (P/V) , In contrast, for microbial cultures, the required gas– according to the van’t Riet equation [8] an increase of liquid oxygen mass transfer coefficient for oxygen has the power input by a factor of approximately 7 should

to be much higher. In these cultures, an oxygen uptake result in an increase of the kLa value by a factor 4. rate of at least 150 mmol/l/h is common and necessary However, in practice, an increase compared with the to compete with traditional stirred tank reactors – it is traditional wave-type bioreactor by a factor of 10 has the basis for reaching a reliable cell density. For such a been observed instead.

volumetric oxygen consumption, the gas–liquid oxygen This is one reason for the accelerated kLa values that -1 mass transfer coefficient has to be at least kLa = 400 h . are achieved in comparison with other wave-mixed sys- Due to the 2D rocking motion in combination with a tems. Another reason for this might be a prolonged film pillow-like design of the culture bag, the CELL-tainer formation and, hence, larger surface area between the

Current expansion process (shake flasks in incubators, wave bioreactors) Vial thaw Culture initiation Seed step 1 Seed step 2 1:4 1:4 40 ml 160 ml

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

Seed step 3 Seed step 4

2.5/10 l1 × 25/50 l 1:10

Batch Perfusion

CELL-tainer® expansion process CELL-tainer® (160 ml–25 l) Vial thaw Culture initiation 1 × 25/50 l 1:4 1:10 40 ml 250 ml SF Bioreactor

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

170 Pharm. Bioprocess. (2013) 1(2) future science group Single-use bioreactors for microbial cultivation Review liquid and gas phase due to the horizontal movement. qualification procedure leads to technical limits in the

The distinct increase of the kLa value in the larger sys- volume size that is applicable. However, SUB design for tem is possibly caused by additional eddy formation. the application of microbial cultivation is already avail- This eddy formation starting from the edges of the bag able on the market. These systems have to be equipped reaches up to the middle of the bag before collapsing with suitable monitoring and control in order to be able (Figure 3). It is appearing at a filling level that is not too to compete with traditional stirred tank systems. low (early collapse of the eddy) and not too large (no At scale-up, mixing times of SUBs are usually af- eddy formation observable). The eddy formation likely fected, since a concomitant increase of power input is increases the power input. However, detailed studies of restricted for mechanical reasons. For example, mixing the fluid dynamics have to be performed. Since the kLa times in the 120 l CELL-tainer are approximately a fac- values at 12 and at 120 l are comparable at both scales, tor of five larger than in the 12 l scale [6]. However, as the growth performance should be as well. long as the characteristic time for pH-control is in bal- The bag is situated in a closed incubator, thus en- ance with the mixing performance, no process-relevant suring very accurate temperature control, which is cru- gradients of pH might be expected. For temperature cial for the application at microbial cultures. Due to control (cooling) no gradients are expected either. the higher volumetric consumption rates, the control of the temperature is much more demanding than at Monitoring SUBs cell line proliferation. For efficient cooling, which is a Since one approach to overcome the mentioned disad- prerequisite for systems suitable for microbial cultures, vantages of SUBs for application to microbial processes the CELL-tainer is supplied with a cooling plate in the is a suitable strategy for appropriate feeding to control bottom of the moving tray. growth (nutrient-limiting fed-batch), monitoring be- comes even more important. Monitoring capabilities of Applications for working volumes above 100 l SUBs are crucial, since sensors connected to these sys- The rocking based SUBs – in mammalian cell culture – tems have to withstand gamma-irradiation and must be are not only applied in laboratories for screening and pro- comparably cheap due to their disposable application. duction of nonclinical material, but also in seed trains. In the main, sensors are applied in two ways: systems A step-wise scale-up in rocking systems is performed up that are cheap and an integral part of the bioreactor to working volumes of 200–500 l. A standard seed pro- and, thus, disposable; and conventional sensors that cess includes at least six steps before sufficient amounts are identical to the ones applied in stirred tank reac- of cells are available to inoculate the production reac- tors made of steel. The requirements of the first group tor (Figure 4). Investigations performed by Shire HGT led to the development of sensors that are often not as (MA, USA) showed that at least three seed steps can be accurate as traditionally applied sensor systems in steel reduced when applying the CELL-tainer SUB, in which stirred tanks. For single-use, dissolved-oxygen and pH so-called ‘expansion blocks’ are used [9]. This offers the sensors based on optical principles are applied, which possibility to create a variable working volume in one are noninvasive. A spot is printed on the foil, on to and the same bag from 0.15–25 l. In this way, handling which reacting material is bound. Most principles rely and risk of contamination is significantly reduced. on phase fluorometry. Light (typically at a wavelength Since mammalian cell culture processes are becom- of 480 nm) is conducted to a spot of fluorescent dye at ing more productive due to advances in cell line engi- the inner side of the bag wall via optical fiber technol- neering and process development, cell culture titers in ogy. The corresponding excited fluorescence (usually a fed-batch processes have increased from 0.05 to over wavelength of 520 nm) depends on the DO and pH and 10 g/l during the last 15 years [3]. Even productivities of is collected through the optical fiber towards a photo- above 25 g/l are reported at the production of a mono- sensor [11] . Thus, the sensor unit is decoupled from the clonal antibody using the XD®-process with the human parts to be sterilized; only the dye is exposed to gamma- ® PER.C6 -cells [10] . As a result, scale-up is restricted to irradiation. Sensor systems for the measurement of dis- volumes of 2000 l, since there is no market demand solved carbon dioxide are currently in use, also relying in the biopharmaceutical industry, which still dictates on an optical determination method (PreSens [Regens- reactor development. burg, Germany] and Polestar Technologies [MA, USA] Bags are always manufactured with material of USP among others). class VI. They are exposed to a qualification process One drawback of this method is the loss of sensitiv- concerning extractables and leachables, as well as steril- ity at the spot due to photobleaching, which restricts ity and integrity. This information is usually available the lifetime. The change in intensity measurement of from the vendors for established systems and from the the fluorescent signal due to gamma irradiation leads suppliers of the developed film materials. This whole to a complex calibration procedure. The second group

future science group www.future-science.com 171 Review Oosterhuis, Neubauer & Junne

generally contains any electrode that can be coupled to Sensors are available for SUBs from Fogale Nanotech a traditional stirred tank system. One example is the and Aber Instruments. The devices can be coupled PadReactor from ATMI where these electrodes are con- with flow-through cells and the electrodes can also be nected via aseptic KleenpakTM connectors from PALL. attached to the bag surface. The sensors are reusable and rely on amperometric DO Noninvasive radio frequency measurements have and potentiometric pH measurement. Since they are been proven to yield a spectrum, which can be coupled not directly in contact with the culture media, presteril- to the estimation of temperature and conductivity [12] . ization is not necessary, which simplifies the calibration By applying multivariate data ana­lysis, these data are procedures. This approach has the advantage of being gained from one single sensor system. The impact of fully compatible to monitoring in steel stirred tank re- environmental conditions on measurements is reduced, actors with respect to accuracy and life time of sensors. proving the suitability of the approach in long-term The CELL-tainer system uses electrochemical pH de- measurements. Nacke et al. describe the application of tection and a micro-amperometric DO measurement. a microwave spectroscopy sensor suitable for continu- While the pH electrode is disposable, the DO sensor ous monitoring of the fermentation media in SUBs. can be reused many times, as only the sensor membrane Between frequencies of 0.3–10 GHz, the permittivity makes up part of the disposable bag. The sensors are and conductivity is determined non­invasively by the in- mounted in small cups at the bottom of the bag, which tegration of a dielectric window as mechanical port [13] . offers the advantage that they are covered with liquid at The determination of process-relevant metabolites, all times, even at low filling volumes and under angled such as glucose, glutamate and lactate, is performed conditions. Since neither electrode relys on fluorescent with biochemical sensors, which comprise of immobi- measurements, they are also characterized by longer life lized enzymes and are distributed gamma-radiated (e.g. times. C-CIT sensors that are available for single-use systems). Beside standard parameters, sensors for impedance While these sensors can be connected directly to the measurements are also applied, with which the capaci- liquid phase, several other microfluidic systems have tance of cells is determined. This provides information also been developed that are mounted outside the reac- on the cell vitality and can be related to cell growth. 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 70 simultaneous determination of glucose, lactate, gluta- Stirred tank BAT1885 60 mine and glutamate. Flow rates were in the range of 0.1 CELL-Tainer® BAT2072 to 100 µl/min. Since these flow-through micro­devices 50 CELL-Tainer BAT2082 are suitable for gamma-irradiation, integration in dis- 40 posable concepts is possible [14] . Other methods com- 30 mon for the online detection of metabolites are near- Induction OD 600 nm 20 IR and Fourier-transformed IR spectroscopy. These 10 methods are similar in that they are not all suitable for a direct combination with single-use systems due to 0 0123456 their complexity and expensive equipment. However, Time (h) these sensors are coupled to bypass pipes (coupled to common cell-retention systems at SUBs) to measure 2500 Stirred tank the cell-free media or, in the case of near-IR, directly 2000 CELL-tainer BAT2082 connected when suitable barriers such as translucent CELL-tainer BAT2072 membranes are installed. Coupling sensors to SUBs is 1500 described by Furey from a practical point of view [15] . The current state-of-the-art and the steady develop- 1000 ment of the sensor technique for disposable systems is

(arbitrary units) 500 broadening the possibilities for improved monitoring Protein production and control, shortening the gap to traditional stirred 0 tank bioreactors and enabling the application for 0123 microbial cultivation. Post-induction hours

Figure 5. Comparison of Escherichia coli growth in a classical stirred Microbial application in wave-mixed SUBs fermenter and the single-use CELL-tainer® bioreactor. As has been stated above, microbial cultures require Reproduced with permission from [22]. more oxygen and more intensive mixing than mam-

172 Pharm. Bioprocess. (2013) 1(2) future science group Single-use bioreactors for microbial cultivation Review malian cell culture processes, despite the application of fed-batch procedures. In wave-mixed bioreactors with 9 -1 STR working volumes of 100 l, kLa values of kLa < 10 h and 8 -1 CT mixing times > 100 s are reported [7]. Typical power 7 consumptions of up to 0.05 W/l for a 2 l wave-mixed 6 bioreactor have been examined. Usually bioreactors for microbial applications are equipped with stirrers 5 transferring 2 W/l and more [8]. 4 OD 620 nm When applying a sparger into the liquid (10 l work- 3 ing volume in a 20 l wave-bag), some improvement 2 can be achieved. In this way, a gas–liquid oxygen mass -1 1 transfer coefficient of kLa = 60 h has been reported. A Wave SUB was applied in this way for the cultivation 0 0 1234567 of Saccharomyces cerevisiae [16] . In a 5 l batch culture, oxygen limitation was not observed until a biomass Time (h) concentration of 8 g/l, due to blending of the inlet gas Figure 6. Growth of Corynebacterium glutamicum in with oxygen. At increased volumes, the oxygen limita- the CELL-tainer® compared with a stirred fermenter. tion was severe and could not be avoided when baffles CT: Cell-tainer; STR: Stirred fermenter. were applied additionally. Courtesy CELLution biotech. Several other applications of bacterial cultivation in targeted than in production processes. A study in a wave-mixed bioreactors are reported in the literature. In WAVE SUB with 5 l cultivation volume described the a batch procedure, a final biomass concentration below growth of an E. coli culture towards the final targeted

2 g/l was achieved in a BIOSTAT CultiBag RM system OD600 of 15. In this range of the cell density, the oxygen when cultivating Escherichia coli before oxygen deple- transfer was sufficient to avoid oxygen limitation[21] . tion [17] . In a nutrient-limited E. coli fed-batch approach Due to the larger kLa values at the CELL-tainer in an optical density (OD) of OD600 = 30 (10 g/l of dry comparison with other wave-mixed bioreactors, it is biomass) was achieved. The applied recombinant E. coli especially useful for the application for microbial cul- RB791 strain expressed a heterologous alcohol dehy- tivation. Several investigations have been performed drogenase. Hence, recombinant protein expression was to prove the performance of the CELL-tainer in the performed successfully in the SUB. At these conditions, cultivation of E. coli. In a study performed by Sanofi- the feed was controlled with the enzymatic substrate re- Pasteur, it has been demonstrated that at a working ® lease method EnBase (BioSilta; Oulu, Finland) [18], volume of 7 l, both growth and protein production while no external feeding was applied. This approach is performance in the CELL-tainer are comparable with very suitable when monitoring systems are not reliable the data obtained in a traditional stirred tank reactor, enough. This methodology can compensate for some as depicted in Figure 5 [22]. In batch cultures, densi- drawbacks existing with current monitoring systems at disposable bioreactors. The application of SUBs for the growth of Coryne- 50 bacterium diphtheriae for vaccine production was inves- 14 tigated by Ullah et al. [19] . In the BIOSTAT CultiBag 40 12 RM reactor, a final cell density of OD = 5 (which can 10 590 30 be considered as approximately 2 g/l of dry biomass) 8 compared with OD590 = 7.3 in an aerated stirred tank 20 6 bioreactor was measured [19] . Hitchcock described the 4 Glucose 10

cultivation of a recombinant Listeria monocytogenes for Cell dry weight (g/l ) 2 concentration (g/l) vaccine production. The product was used for Phase II 0 0 clinical trials. A BIOSTAT CultiBag RM bioreactor 01020304050 was applied due to easier validation in comparison to Time (h) a traditional steel bioreactor. When the filling volume was reduced to 5 l, a sufficient gas–liquid oxygen mass 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 transfer at a final OD of 12 was achieved [20]. 600 and straight line) and 120 l (open symbols and dashed line) in the CELL-tainer® A suitable further application of wave-mixed SUBs is single-use bioreactor. The cell dry weight is indicated as circles, the glucose the utilization for inoculum cultivations. This applica- concentration as triangles. tion is beneficial, while a smaller cell density is usually More information available from [6].

future science group www.future-science.com 173 Review Oosterhuis, Neubauer & Junne

ties of OD600 = 60 were obtained and in the fed-batch of 120 l [6]. In this study, a genetically transformed

mode even OD600 = 90 was achieved. This study proves E. coli B21-Gold strain (Agilent Technologies; DE, the potential of the application of SUBs for microbial USA) was used that encoded a maltogenic amylase. The cultures. cultivation was running in a nutrient-limiting fed-batch The CELL-tainer SUB is applied in a seed train for mode. At a working volume of 12 l and only after 32 h,

production of lysine by Corynebacterium glutamicum. a final OD600 of over 130 was reached. At the tenfold Usually, during first steps in such a large-scale pro- scale a similar biomass yield was obtained, proving the cess, shake flasks are applied. As the working volume successful scale-up of the process (Figure 7). of a shake flask is limited to approximately 1 l, several flasks have to be pooled to inoculate one large fermenter Microbial application in stirred tank SUBs & (>10 m3). Both pooling of the different flasks and flask- orbital shakers to-flask variability may lead to problems in such a seed Due to the beneficial height-to-diameter ratio and less train. Compared with shake flasks, a bioreactor reduces required ground space, bags with larger volumes than handling and provides a controlled preculture (pH and 500 l are available for stirred SUBs. Usually, these re- DO control). The seed process can also be performed actors are equipped with several blade impellers and in a standard stirred (autoclavable or in situ sterilizable) microspargers such as the XDR from XCellerex (GE fermenter. However, for this setup, a more complex Life Sciences) and the BIOSTAT CultiBag STR from infrastructure is needed, fermenters demand cleaning Sartorius. Galliher et al. report a 50 l cultivation of (manual operation) and there is less flexibility. In com- E. coli in a XDR-50 SUB [25,26]. In this process, a cell

parison with a traditional stirred tank reactor, growth density of OD600 = 120 (dry cell weight of 40 g/l) was in the CELL-tainer shows comparable results (Figure 6). achieved. In addition, Pseudomonas fluorescens was As discussed in the introduction, the application of cultivated in the XDR. In this case, a dry cell weight SUBs for marine cultures has benefits in comparison of over 100 g/l was obtained. In this study, the process with the cultivation in steel stirred tank reactors. Re- performance was described as similar to cultivations cently, own studies of the CELL-tainer technology for in conventional nondisposable stirred tank reactors the cultivation of heterotrophic microalgae in marine [24,25]. When a sufficient control strategy is applied, media has been proven to be sucessful. Although nu- similar yields as those obtained in stainless stirred trient-limitation cannot be processed in this case, the tank reactors can be achievable in microbial cultiva- gas transfer was sufficient to achieve a cell dry weight of tions using SUBs. Based on the maximum oxygen over 45 g/l at maximum growth rates of 0.05 h-1. Lehm- transfer capacity available in the BIOSTAT CultiBag an et al. performed a study in which the suitability of RM and BIOSTAT Cultibag STR, the medium feed SUBs for application with phototrophic algae was in- is adapted to a linear feed instead of an exponential vestigated [23]. The different systems that were observed feed for a wave-mixed BIOSTAT CultiBag RM and were a BIOSTAT CultiBag RM, which was equipped an exponential feed for a stirred BIOSTAT CultiBag

with red and white LEDs, the wave-mixed AppliFlex STR was applied. An OD of up to OD600 = 140 was (Applikon Biotechnology) with white light LEDs, and achievable at E. coli cultivations [27]. the orbital shaken CultiBag RM on the Multitron Cell The orbitally shaken bioreactor SBX from Kuhner shaker (Infors HT; Bottmingen, Switzerland) with has no inlet parts due to the fact that the whole reactor white fluorescent tubes. In all systems, similar cell den- is shaken as a consistent scale-up from culture flasks -1 sities were achieved, such as in stirred, helical tubular [28]. Although kLa values are approximately 10 h , and an airlift photobioreactor. which is sufficient for achieving satisfying results Another field of application of SUBs for microbial with mammalian cell cultures [29], our own studies cultivation is in anaerobic processes. The bacterium have also yielded good results at cultures with higher Eubacterium ramulus was used as model organism in oxygen demand, such as heterotrophic microalgae. a culture of a working volume of 10 l in a BIOSTAT Another study reports the successful cultivation of CultiBag RM SUB [24]. In this culture, a final wet bio- transgenic tobacco cells. After 6 days of cultivation, mass concentration of 60–70 g/l was achieved, which is a dry cell weight of 12 g/l was achieved at a volume comparable with results obtained in steel stirred tank of 10 l [30]. reactors. The characterization of these reactors, with respect to the power input and gas–liquid oxygen mass trans- Scale-up of microbial cultivations in the fer, requires the consideration of further constraints CELL-tainer bioreactor than for directly stirred SUBs. This has been ac- Recently, an investigation showed the potential of complished at the laboratory scale [31] and is ongoing growing E. coli cultures in a larger cultivation volume for larger orbital shakers, when, for example, liquid-

174 Pharm. Bioprocess. (2013) 1(2) future science group Single-use bioreactors for microbial cultivation Review film formation and the concomitant increase in the processes. This results in a significantly higher (factor surface-to-volume ratio has to be considered. 10–20) K La. The combination of intelligent software sensor control strategies and currently improving (dis- Conclusion posable) sensors will lead to a reduction of drawbacks. The advantages in applying SUBs for microbial pro- For example, the correct settings for nutrient-limiting cesses are equal to those for mammalian processes. As fed-batch procedures can be achieved by appropriate microbial processes usually running for 1–5 days, time sensor-based control. Limitations of sensor application for cleaning and sterilizing of the bioreactor can exceed such as correct biomass determination can be circum- 20% of the process time. Hence, besides complex in- vented by these strategies, when affordable and – even frastructure, more fermentation capacity is also needed more important – reliable and robust sensors for dispos- when applying steel bioreactors compared with single- able bioreactors are widely applicable. use systems. For laboratory-scale fermentations, usually autoclavable glass fermenters are applied. Although the Future perspective infrastructure is less demanding, time needed to clean The benefits of using SUBs in biopharmaceutical pro- and to autoclave such glass fermenters is laborious. A cessing are significant. Costs of production can be re- yearly cost saving of €50,000 can easily be achieved duced, and due to a greatly simplified infrastructure, when using a single-use fermenter (10 l working vol- industry can shorten the timelines of getting products ume, compared with an autoclavable fermenter) [32]. into the clinical trial phase, which is a key driving factor. To date, presently available SUBs are less suitable The availability of SUBs for microbial cultivations for cultivation of microbial cultures. Due to limitation widens their potential, not only in biopharmaceutical in mass transfer and mixing, cell densities are limited processing, but also as a preculture bioreactor for bulk in those SUBs that are designed for mammalian cell fermentations. It is expected that in the next 5–10 years, culture. A comparison of different techniques is not the bioreactor landscape will be covered with many more easy to perform, since only the gas transfer and mixing single-use systems. times can be compared applying standard techniques. Further considerations such as the power input are not Disclaimer trivial to perform and demand time. Some work has al- The views expressed in the article are the author’s own and do not ready been performed for the BIOSTAT CultiBag RM necessarily reflect the views of Future Science Ltd. wave-mixed reactor [5]. However, for other systems such as the SBX or the CELL-tainer, research is still ongoing. Financial & competing interests disclosure The CELL-tainer SUB is available for working vol- NMG Oosterhuis is CTO/CSO of CELLution Biotech BV, being the umes up to 150 l. Based on growth data of E. coli and C. company who developed the CELL-tainer®. The authors have no other glutamicum cultures, it can be concluded that this reac- relevant affiliations or financial involvement with any organization tor type is very suitable for microbial cultivation. Due or entity with a financial interest in or financial conflict with the sub- to a 2D rocking motion, the turbulence in the liquid ject matter or materials discussed in the manuscript apart from those is intensified and dissipated power per volume is com- disclosed. No writing assistance was utilized in the production of parable with standard stirred fermenters for micro­bial 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.

future science group www.future-science.com 175 Review Oosterhuis, Neubauer & Junne

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