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2.19 Disposable Bioreactors NMG Oosterhuis, CELLution Biotech BV, Assen, The Netherlands T Hudson, Genentech, Inc., Oceanside, CA, USA A D’Avino and GM Zijlstra, DSM Biologics, Groningen, The Netherlands A Amanullah, Genentech, Inc., Oceanside, CA, USA
© 2011 Elsevier B.V. All rights reserved.
2.19.1 Introduction 249 2.19.2 Types of Single-Use Bioreactors with Disposable Bags 250 2.19.2.1 Rocking-Type Bioreactors 251 2.19.2.1.1 Wave Bioreactor™ 251 2.19.2.1.2 CELL-tainer® bioreactor 252 2.19.2.2 Application Examples of Cell Culture in Rocking-Type Bioreactors 254 2.19.2.2.1 Cultivation of CHO cells in various rocking-type bioreactors 254 2.19.2.2.2 Fed-batch culture of PER.C6® – cells in the CELL-tainer® single-use bioreactor compared with a stirred bioreactor 255 2.19.2.3 Stirred Single-Use Bioreactors 256 2.19.2.4 Application Examples of Stirred Single-Use Bioreactors 257 2.19.2.4.1 CHO culture in single-use bioreactors 257 2.19.2.4.2 PER.C6® culture in single-use bioreactor (fed-batch) 257 2.19.2.4.3 PER.C6® culture (XD® process) in single-use bioreactors 260 2.19.3 Conclusions 260 References 261 Relevant Websites 261
Glossary CELL-tainer® Single-use bioreactor (SUB) with pillow-shaped bags applying a two-dimensional rocking motion of the bag, resulting in a high oxygen mass transfer. chinese hamster ovary (CHO) cells Mammalian cell line (CHO cells) widely applied as host cell for the production of biotherapeutic proteins or antibodies. oxygen mass transfer coefficient (kla) Value expressing the capability to transfer oxygen from the gas phase to the liquid phase. PER.C6® cells Human cell line widely applied as host cell for the production of biotherapeutic proteins or antibodies and proprietary to Crucell, the Netherlands.
SUB Single use bioreactors with a tank liner bag equipped with top- or bottom-driven disposable stirrers (as for example introduced by Hyclone). single-use bioreactor Bioreactor applying a presterilized bag, which is only used once and disposed after the process. wave Bioreactor™ SUB with pillow-shaped bags applying a rocking type of motion of the bag. XD®(eXtreme Density) process Proprietary DSM Biologics’ XD® technology has been developed to drive yield improvements in mammalian systems and to push the cell density within the bioreactor to maximum productivity.
2.19.1 Introduction
With the increasing number of therapeutic candidates, such as monoclonal antibodies, biotherapeutic proteins, and vaccines entering early-stage process development, biopharmaceutical companies are increasingly looking at innovative solutions to deliver the pipeline. Time to market, cost-effectiveness, and manufacturing flexibility are key issues in today’s competitive market, where several companies are working on therapies for similar clinical indications, and these must all be achieved while maintaining the desired product quality.
Traditionally, glass vessels or stainless-steel tanks have been used at laboratory and pilot scales for process development and production of research grade, toxicology, and phase 1 clinical material. Stainless-steel tanks dominate large-scale manufacture (1000–25 000 l) of biotherapeutics. However, the use of fixed plant equipment is costly, requiring long lead times for installation of the tanks and supporting infrastructure and qualification. There is also a high burden from validation efforts related to sterility and cleaning, as well as maintenance. The risk for cross-contamination in standard (steel or glass) equipment leads to strict rules for cleaning and validation.
In the last decade, the use of disposable technologies has increased considerably in the biopharmaceutical processes. Originally used in the biotechnology field for the processing of blood and blood products, disposable technologies expanded into the
249
250 Bioreactors – Design
bio-pharmaceutical arena for medium preparation and storage of buffers. Although already mentioned in a publication of Kybal and Sikyta in 1987 [1], the first real breakthrough of a single-use reactor for cell culture was realized by Singh in 1999 [2]. Since then, various types of disposable technologies have been introduced, all with their specific benefits and drawbacks compared to the traditional stirred bioreactors.
Disposable technologies offer significant advantages over traditional fixed plant equipment, not only at pilot scales of operation, but also for laboratory purposes, and even in a Good Manufacturing Practice (GMP) production environment. They can be introduced rapidly into a laboratory or manufacturing facility, as installation, qualification, and personnel training efforts are minimal, and few utilities are required. The product contact portions of disposable systems are provided clean and presterilized; therefore, there is no need for clean-in-place (CIP) or steam-in-place (SIP) operations, and extensive validation studies for sterilization and cleaning can be eliminated. These systems offer increased reliability due to their onetime use. In addition, they can increase plant capacity and flexibility by reducing turnaround time (especially in the event of contaminations), decreasing setup time, and demanding a smaller footprint due to significantly reduced piping, valve, and instrumentation requirements. Operational flexibility is essential due to changing priorities in development. Disposable systems allow continuous improvement and integration of new technologies such as online monitoring systems to occur more rapidly because implementation of design changes can be made faster and cheaper with disposables than with traditional stainless steel and glass equipment. In addition, facility changeover time and effort can be reduced, especially in GMP applications. It should be noted that disposable systems do carry certain risks that are not in common with traditional systems. One such example is the possible dependence on a single supplier and the risk of a stockout. As many of the disposables are protected with patents, dual sourcing can be difficult or impossible. Overall, however, the advantages of disposables often outweigh the risks and lead to significantly lower capital costs and lower resource (personnel) requirements, which is a key factor for both large and small companies alike.
Cell culture processes are becoming more productive due to advances in cell line engineering and process development, which in turn allows the use of smaller scales. In the past 15 years, cell culture titers in fed-batch processes have increased from 0.05 to over 10 gl−1 today [3]. As a result, smaller bioreactors are gaining popularity, and this lends itself to implementing disposable technologies. The use of disposables facilitates the production of gram quantities of research-grade material, scale-up and the handling of multiple products in a Good Laboratory Practice (GLP) or GMP facility for the manufacture of toxicology or even phase 1 and phase 2 material. For selected products with lower market requirements, production even at a commercial scale is possible in single-use equipment, adding much more flexibility to an operating plant.
Disposable technologies in bioprocessing cover a wide range of components such as bioreactors, tubing/pipes, pumps, filters/ membranes, valves, sampling devices, bottles, bags for media and buffer preparation, and sensors to name a few. The applications range from culture cultivation, purification (cell separation, buffer bags, in-process pool containment, and prepacked chromatography columns), and bulk drug storage. For screening, clone optimization, and medium optimization, disposable medium and high-throughput equipment are available as well. This might vary from a disposable version of the traditional shake flask to high-throughput equipment such as the SimCell™ microbioreactor with a volume of 1 ml (Seahorse Biosciences, www.seahorsebio.com). In all of these areas, disposables are gaining popularity, but there may be additional drivers behind the change toward disposables than those discussed here regarding laboratory to production-scale bioreactors.
The focus of this section is on single-use bioreactors for culture processes with a starting volume of 1 l.
2.19.2 Types of Single-Use Bioreactors with Disposable Bags
Key in the use of single-use bioreactors is the application of disposable bags that are available with or without integrated sensors; equipped with connections for feed, inoculum, and sampling; and with inlet and outlet gas filters. These bags are presterilized using gamma irradiation at levels between 25 and 50 kGy, which ensures full sterility.
One of the challenges for using these presterilized bags is ensuring proper mixing and mass transfer as well as proper process measurement and control that is comparable to that of traditional stirred bioreactors. This is especially the case for more demanding processes such as a high-cell-density perfusion process (cell densities of 50 � 106 cells ml−1 and above) or fungal, yeast, or bacterial processes (that require oxygen transfer levels ≥150 mmol l−1 h−1), of which many are highly viscous as well.
Today, single-use bioreactors are available at various scales from the laboratory and pilot scales (1–100 l) up to manufacturing scale (1000–2000 l). The incorporation of disposable bag technology is the core of their design and enables mixing, mass transfer, and process parameter control (airflow rate, gas mixing, temperature, dissolved oxygen (DO), and pH) similar to that of conventional stirred tanks.
The bags are generally manufactured with class VI US Pharmacopeia (USP) materials. For many years, liners and bags composed of these materials have been used for media makeup, storage, and transfer; hence, cultivating cells inside such materials should not be viewed as a substantial risk. Prior to the implementation and use of single-use bioreactors, they must be qualified in terms of extractable/leachable (e.g., total organic content or TOC) material and vapor transmission from the material of construction of the bags used. Integrity and sterility qualifications must also be completed. These qualifications can be performed using standard methodologies such as the use of model solvents and media hold tests under processing conditions. This is generally done with the complete bag design, which includes the bag, fittings, and tubing. The extractable/leachable information is generally available from the vendors for more established systems. In addition, single-use bioreactors have to be characterized in terms of their mixing,
Disposable Bioreactors 251
Table 1 Overview of commercially available disposable bioreactors
Reactor type Working volume
Type of bag
Type of mixing Supplier Patent
kla (hr −1) Further information
Wave Bioreactor™ 1–200 l Pillow Rocking GE Healthcare US6190913 < 10 www4.gelifesciences.com Cultibag RM 1–100 l Pillow Rocking Sartorius Stedim EP1778828 < 10 www.sartorius-stedim.com
Biotech Appliflex 1–25 l Pillow Rocking Applikon < 40 www.applikon-bio.com
CELL-tainer® 0.2–25 l Pillow 2D Biotechnology
CELLution WO2008153401 > 200 www.cellutionbiotech.com rocking Biotech
Cultibag STR200 50–200 l Tankliner Stirred Sartorius Stedim DE102006021984 < 40 www.sartorius-stedim.com Biotech
Single-Use 50–1000 l Tankliner Stirred Thermo-Fischer US2005/0239199 < 40 www.thermo.com Bioreactor (SUB) (Hyclone)
XDR single-use 40–2000 l Tankliner Stirred XCellerex WO2005118771 < 20 www.xcellerex.com bioreactor
Nucleo single-use 50–100 l Square Paddle ATMI/Pierre WO2007134267 < 20 www.atmi-lifesciences.com bioreactor 3D Guerin
Shaking bioreactor < 200 l Tankliner Orbital Kuhner/ www.kuhner.com shaker ExcellGene
CellMaker Regular 1–50 l Bubble Rotating Cellexus US2007148726 < 10 www.cellexusinc.com column sparger
oxygen and CO2 gas–liquid mass transfer, and process control capabilities (response time and accuracy) for temperature, DO, pH, and possibly other parameters. However, challenges still exist in cultivation of cholesterol-dependent cell lines such as NS0 [4].
The most commonly used single-use bioreactors are the Wave (rocking) type bioreactors (GE Healthcare, Sartorius, and Applikon) and the stirred bioreactors (Thermo-Fisher, Sartorius, and XCellerex). These types will be discussed in more detail with some examples of operation. An interesting new type of rocking bioreactor is the CELL-tainer® by CELLution Biotech, which is based on a two-dimensional movement and results in much higher mass transfer values than the classical rockers, and can therefore be applied for microbial applications as well. Table 1 provides an overview of commercially available disposable bioreactors.
2.19.2.1 Rocking-Type Bioreactors
2.19.2.1.1 Wave Bioreactor™ One of the first single-use bioreactors introduced for cell culture applications was by Wave Biotech and was coined the Wave Bioreactor™ [2]. The Wave Bioreactor™ (Figure 1) consists of a disposable bag, which contains cells and media, placed on a heated rocker; headspace aeration is used to inflate the cellbag, and the rocking motion ensures mixing and mass transfer.
Figure 1 Wave Bioreactor™ example. Photo courtesy of Genentech Inc.
252 Bioreactors – Design
DO and pH are usually measured by optical probes. Gas blending to maintain DO and pH is supplied using mass flow controllers and integrated controllers. The manufacturers of this system, GE Healthcare Bioscience Bioprocess Corp., (Somerset, NJ, USA), claim suitability for a wide variety of cell types, and have manufactured units with up to 500 l working volume. However, mechanical issues have been noted at larger scales where repeatedly stopping and starting a 500- l wave of liquid can impose a major strain at the motor and rocker assembly. In addition, they have also produced a system capable of operation under perfusion conditions. This system involves the use of a cellbag specifically designed for perfusion applications that contains an internal polyethylene filter. The floating 7-μm-pore-size polyethylene filter is approximately 100 cm2 and allows the continuous withdrawal of cell-free spent medium from the system, which is replaced with fresh medium to maintain culture volume (at 10 and 100 l volumes [5]) . Filter fouling, a common problem in membrane-based cell retention devices, is reduced due to the wave motion of culture fluid across the filter. Perfusion control, based on constant culture weight, relies on a load cell integrated into the Wave Bioreactor™ platform.
Oxygen transfer coefficients of 10–30 h−1 have been reported by various authors [6]. Mixing times are in the order of magnitude of 2–3 min for scales, up to 100 l. Especially at lower rocking speeds (< 20 rpm) and at larger scales (> 100 l), the mixing time increases significantly (up to 5 min). From a simple regime analysis at the large scale, one may conclude that the Wave-type bioreactors are working in a so-called mixed regime where mixing time and mass transfer are in the same order of magnitude, which may lead to gradients of oxygen and CO2. As temperature control is ensured by an electrical heating device directly under the bag, temperature gradients may also occur, especially in the larger systems. Scalability of the system is not obvious as demonstrated by Eibl and Eibl [7], showing very different mixing times, mass transfer values, power inputs, and Reynolds numbers at the various scales.
Publications exist on batch cultivation of mammalian, insect, yeast, and plant cells within the Wave Bioreactor™ [8–10]. However, little literature exists regarding the use of this system for more intensive processes, such as fed-batch or perfusion operation. Pierce and Shabram [11] used Wave bioreactors coupled to an external hollow fiber 0.2-μm pore size microfiltration cartridge for a perfusion culture of a CHO-based cell line for the production of fusion protein at scales up to 500 l. Tang and Hamel [12] reported the use of a 1-l perfusion Wave Bioreactor™ for the production of a immunoglobulin G (IgG2) monoclonal antibody using hybridoma cells.
Most literature report cell densities of up to 6–7 � 106 cells ml−1 in Wave-type bioreactors for various cell lines, although also higher cell densities up to 15 � 106 cells ml−1 have been reported. Not only do reactor properties influence the cell densities, but certainly also types of cell line, feed profile, and culture conditions in general.
A number of similar wave-like systems, operating on similar principles, have been recently introduced, including those from Sartorius (Cultibag) and Applikon (Appliflex). Both systems from Sartorius and Applikon are equipped with invasive fluorescence-based optical DO and pH sensors (an improvement over the current sensors used in the Wave Bioreactor™). Probe drifts are common due to photo-bleaching effects and generally can be minimized for the duration of typical cultivation times by decreasing the frequency of measurement to every minute or longer.
2.19.2.1.2 CELL-tainer® bioreactor Based on a rocking motion and the application of a pillow-shaped bag, the CELL-tainer®, due to a two-dimensional rocking motion (in vertical and horizontal directions), results in a much higher mass transfer [13] (Figure 2).
High mass transfer ensures the potential of supporting higher cell densities, better stripping capacity of CO2, and application potential in both microbial and fungal cultures.
Besides improvement of the mass transfer, this bioreactor offers a removable segmentation of the bags; one can start even at volumes <250 ml and expand the culture in the same bag up to working volumes of 15 l. The sensors (traditional electrochemical pH and polarographic DO electrodes, but in a disposable format) are mounted at the bottom [14] of the bag and placed in small cups, which guarantees proper process control even under shaking conditions with low volumes.
Temperature control is ensured by placing the moving bag in an incubator instead of the use of a heating blanket directly under the bag. For cooling (needed when microbial cultures are performed), the system is equipped with integrated cooling plates in the moving tray.
To place the bags in an easy way, the tray can be taken out of the incubator as a drawer. Due to the two-dimensional movement, the mass transfer in the CELL-tainer® single-use bioreactor is much higher compared to
a traditional Wave-type bioreactor. Investigation of the mass transfer, using the dynamic method (tap water, 20 oC), shows that the CELL-tainer® equipment covers a wide range of mass transfer values (Figure 3).
In most mammalian cultures, the mass transfer for oxygen is sufficient to support high cell densities, at least in stirred bioreactors. To enhance oxygen transfer, in stirred bioreactors, usually microspargers are applied and, in addition, oxygen-enriched air is used. The exchange of CO2 in both stirred and in wave-type single-use bioreactors might be limited due to a lower mass transfer coefficient (kla for CO2 =0.89 � kla for O2), but especially also due to limitations in stripping efficiency. As the mass transfer coefficient in the CELL-tainer® bioreactor is far higher than that in wave-type and stirred single-use bioreactors, the liquid-phase CO2 concentration is in equilibrium with the gas-phase CO2 concentration. This results in less CO2 buildup in the liquid phase, which results in lower base addition.
Eibl and Eibl [15] report a specific power input P/V = 50 Wm−3 (at 20 rpm rocking speed) for a Wave BioreactorTM resulting in a kla = 10h−1 (at a volume of 1 l). In the CELL-tainer® (at 10 l) also running at 20 rpm, a power input of P/V = 360 Wm−3 is reached, resulting in a kla = 100 h−1 (non-published results). Following the van ‘Riet [16] equation for the relation between power input and oxygen transfer coefficient, one may conclude that the mass transfer in the CELL-tainer® is more efficient than in the Wave
Disposable Bioreactors 253
Figure 2 CELL-tainer® single-use bioreactor. Photos courtesy CELLution Biotech.
Mass transfer coefficient (kla) as function of rotational speed at different angles
0 10 20 30 40 50 60
350
300
250
200
150
100
50
0
k l a
(h –1
)
16°
12°
8°
Rotational speed (rpm)
Figure 3 Mass transfer in a prototype CELL-tainer® bioreactor (15 l water, 20 oC). Data courtesy of CELLution Biotech.
kla vs. airflow rate 200
175
150
125
0 0.5 1 1.5 2 2.5
Air overlay flow rate (lpm)
k l a
h–1
100
75
50
25
0
kla vs. RPM
18 rpm
30 rpm
50 rpm
0
20
40
60
80
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160
180
200
k l a
h –1
0 10 20 30 40 50 60
RPM
254 Bioreactors – Design
Figure 4 Oxygen mass transfer coefficient as a function of airflow rate and rocking speeds. Data courtesy of Genentech, Inc.
bioreactor, which explains high kla values as observed in the CELL-tainer® bioreactor. (As kla is a function of (P/V)0.7, an increase of the power input with a factor 360/50 = 7.2, should result in an increase of the kla by a factor of 4. However, an increase with a factor of 10 has been observed.)
Using the classical dynamic kla measurement method, the kla in cell culture medium was experimentally determined under various rocking and air overlay flow rate conditions. Values of up to 175 h−1 were measured at the highest rocking rate tested (Figure 4). No influence of the airflow rate was observed, which can be explained by the fact that surface aeration is applied. As can be expected, there is a strong influence of the rocking rate (power input). For cell culture processes, a rocking rate of maximal 30 rpm is advised, resulting in a kla of 60h−1 in a cell culture medium.
The significantly higher mass transfer that can be achieved in the CELL-tainer® bioreactor (kla > 300 h−1) opens the possibility of applying single-use equipment to microbial cultivations as well.
2.19.2.2 Application Examples of Cell Culture in Rocking-Type Bioreactors
2.19.2.2.1 Cultivation of CHO cells in various rocking-type bioreactors Due to their increasing use throughout the bioprocessing industry, three of these rocking-type bioreactors were evaluated in the lab and compared. The bioreactor systems were tested at the 10-l scale in a head-to-head comparison with a traditional 10-l stirred tank (Applikon, Foster City, CA, USA) using CHO cells producing an IgG1 recombinant protein. The rocking systems were a Wave 20/50 (GE Healthcare, Piscataway, NJ, USA), Cultibag RM (Sartorius Stedim Biotech, Bohemia, NY, USA), and CELL-tainer® (CELLution Biotech, Assen, The Netherlands). The initial cell culture process conditions for the production stage were 37 °C and pH 7 and shifted lower to 33 °C and pH 6.8 on day 2. The 10-l bioreactor was operated at process conditions similar to the rocking system. Samples were taken daily and used for titer, cell growth, viability, osmolality, and metabolite analysis. Although minor differences were observed in specific measurement profiles, like lactate rising late in the batch in the Wave 20/50 or lower growth and glucose consumption in the Cultibag, taken as a whole, the data (Figure 5) suggest the different bioreactor systems are roughly equivalent when used with standard conditions for CHO cultures.
Titer (mg l–1) %Viable
Run time (days)Run time (days)
%PCV Glucose (g l–1)
Run time (days) Run time (days)
Glutamine (mM) Glutamate (mM)
Run time (days)Run time (days)
NH4+ (mM) Lactate (g l–1)
Run time (days) Run time (days)
50
60
70
80
90
100
0
1000
2000
3000
0 2 4 6 8 10 12 14
10 l Cultibag 10 l Celltainer 10 l Wave 10 l Stirred tank
0 2 4 6 8 10 12 14
0
1
2
3
4
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6
0 2 4 6 8
10 12 14
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
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0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
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Disposable Bioreactors 255
Figure 5 Comparison of titer, culture growth, and metabolic profiles for Wave-type bioreactors to a stirred-tank bioreactor.
2.19.2.2.2 Fed-batch culture of PER.C6® – cells in the CELL-tainer® single-use bioreactor compared with a stirred bioreactor The PER.C6® fed-batch process was downscaled in rocking-type bioreactors, which are easier to control and require limited operator skills and infrastructure compared to stirred bioreactors. Due to the high mass transfer rate necessary to support high-cell-density fed-batch PER.C6® cells, the CELL-tainer® single-use bioreactor was chosen. Two different CELL-tainer® working volumes (4 and 10 l) were tested, and the performance was compared to a conventional 5-l stirred tank bioreactor (4 l working volume; Sartorius). The same feeding strategy was used. The 5-l bioreactor was connected to a DCU3 (Sartorius) and equipped with micro-sparger, using a gas mix station with air and oxygen to actively control the DO tension, while the DO tension in the CELL-tainer® was not controlled. A flow of air, enriched with CO2, was applied through the headspace. Rocking speed and angle were adjusted during the runs, according to cell growth and viability.
PER. C6® cell densities as high as 20–25 � 106 cells ml−1 could be achieved in the CELL-tainer® bioreactor operated at two different working volumes, resulting in an exponential growth phase very comparable (or even somewhat better; 10 l volume process) to the stirred bioreactor (Figure 6). Beyond generating adequate mass transfer for supporting high biomass concentrations,
30 Viable cell density (*10E6 cells ml–1)
100% Viability (%)
25 80%
20 60%
15 40%
10
5 20%
0 0 2 4 6 8 10 12 14 16 18 20
0% 0 2 4 6 8 10 12 14 16 18 20
Time (days)Time (days)
4 L-Stirred bioreactor 4 L-CELL-Tainer 10 L-CELL-Tainer 4 L-Stirred bioreactor 4 L-CELL-Tainer 10 L-CELL-Tainer
pCO2 (%) pH
6
6.4
6.8
7.2
7.6
8
0
2
4
6
8
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14
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 Time (days) Time (days)
4 L-Stirred bioreactor 4 L-CELL-Tainer 10 L-CELL-Tainer 4 L-Stirred bioreactor 4 L-CELL-Tainer 10 L-CELL-Tainer
7 Product titer (g l–1)
8 Product titer vs. IVC
6 7
5 6
2
3
4
2 3 4 5
1 1
0 0
2 4 6 8 10 12 14 16 18 20 0 0
50 100 150 200 250 300 350 Time (days) IVC (*106 cells*m l/days)
4 L-Stirred bioreactor 4 L-CELL-Tainer 10 L-CELL-Tainer 4 L-Stirred bioreactor 4 L-CELL-Tainer 10 L-CELL-Tainer
256 Bioreactors – Design
Figure 6 Comparison of titer, culture growth, and pCO2 for the CELL-tainer® bioreactor to a classical stirred-tank 4-l bioreactor with PER.C6® cells.
the two-dimensional rocking motion of the CELL-tainer® also provided the stripping capacity required to keep pCO2 below inhibitory levels. IgG productivity also equaled that normally observed in stirred bioreactors. Specific productivities in the CELLtainer® were comparable or, at high IVCs, even higher than the conventional bioreactor. Such a process makes it possible, and relatively straightforward, to fast track supply of representative product (60–80 g) for pre-clinical applications and downstream processing development.
2.19.2.3 Stirred Single-Use Bioreactors
The HyClone SUB (Figure 7), sold through Thermo Fisher Scientific, Inc. (Fremont, CA, USA), is intended as a retrofit product to replace the stainless steel bioreactor vessel in existing bioreactor skids rather than as a complete turnkey bioreactor system with integrated control systems. Alternatively, the HyClone SUB can be integrated using a number of customizable control systems such as Delta V (Emerson, St. Louis, MO, USA). The current line of products consists of customizable 50, 100, 250, 500, and 1000 l maximum working volume units. XCellerex (Marlborough, MA, USA) also markets similar scale units and recently introduced a 2000-l unit. ATMI markets the Nucleo range (up to 1000 l scale).
Disposable Bioreactors 257
Figure 7 A 250-l HyClone SUB connected to a DCU3 control system. Photo courtesy DSM Biologics.
In the Hyclone SUB’s, the disposable bags are mounted inside a permanent stainless steel jacket and secured by clips. Heat transfer is provided by an electrically heated jacket or customizable heat exchangers. The bags, made of class VI USP materials, are fitted with presterilized air inlet and vent filters (with optional heaters to prevent condensation), ports for media/base addition and sampling. A single 45º pitched blade impeller made of molded polyethylene is linked to the seal/bearing assembly by C-flex tubing, which forms the contact material to the shaft. Aeration can be provided via either overlay or sparger. In the latter case, gas is provided by an integrated gas-permeable disk or sparge tube at the bottom of the bag. Overpressurization of the bag can be prevented using disposable pressure transducers linked to a control system. The sealing/bearing assembly links with the motor and allows impeller rotation while maintaining bag integrity. The XCellerex single-use stirred bioreactors are equipped with magnetic drives, mounted at the bottom of the bioreactor, while the ATMI Nucleo range applies a shaking paddle as a mixing device.
The bags can be used with presterilized conventional 12-mm sensors for pH and DO that can be inserted in a sterile manner into the bag using novel quick-connects. Those sensors must also be connected to a control system for data gathering and process control.
Mass transfer in the stirred SUBs is typically restricted to kla = 7–15 h−1, where mixing times are in the order of magnitude of 45–90 s [15]. Due to the low mass transfer capacity and relatively proper mixing, no gradients in oxygen may be expected. In addition, the distribution of CO2 in the vessel will be rather uniform. Increase of mass transfer is possible by the introduction of micro-spargers or a gas-permeable disk creating small bubbles. As with standard stirred bioreactors, the stripping capacity of CO2
is restricted. Due to the low kla values observed in such type of equipment, the SUBs are less suitable for microbial or fungal processes.
2.19.2.4 Application Examples of Stirred Single-Use Bioreactors
2.19.2.4.1 CHO culture in single-use bioreactors The HyClone system has been successfully used to grow CHO cultures producing IgG1 recombinant proteins at the 250-l scale that were equivalent in performance to the 2- and 2000-l stainless steel bioreactor. The HyClone SUB was controlled by a DeltaV-based BioNet system (Broadley James, Irvine, CA, USA). A number of customized modifications were made to the standard bag to address shortcomings in the original design. To reduce foam-outs and avoid bag rupture, the vent-filter tube was extended and branched into dual vent filters, and pressure transducers were also added to the system. The jacket was also modified from an electric blanket to a liquid jacket and a heat exchanger, allowing for heating and cooling. The 2-l (Applikon, Foster City, CA, USA) and 2000-l bioreactors were operated at process conditions similar to the HyClone system. Samples were taken for cell titer, cell growth, viability, osmolality, and metabolite analysis. Additionally, material from a batch at the 2-, 250-, and 2000-l scales was processed through a ProteinA purification step and analyzed for product quality attributes, including high- and low-molecular-weight species, percentage of charge variants, and glycoform distribution. Excellent scale equivalency was obtained as shown in Figures 8 and 9.
2.19.2.4.2 PER.C6® culture in single-use bioreactor (fed-batch) HyClone SUB systems were successfully used to scale up a high-yielding fed-batch process using an IgG-producing PER.C6® cell line. The process, initially developed on 5-l glass bioreactors (Sartorius) by PER.C6® R&D Centre Percivia (Cambridge, MA, USA), was scaled up stepwise to a 50-l HyClone SUB, and, eventually, to a 250-l SUB, at DSM Biologics (Groningen, the Netherlands). The same conditions of temperature, pH, DO, and pCO2 control and the same feeding strategy were used. The same power input per volume was applied to all the scales.
Product concentration (mg l–1) 2
1.6 1.2 0.8 0.4
0
2-l 250-l 2-kl
0 2 4 6 8 10 12 14 16 18 Run time (days)
NH4+ (mM) 10 8 6 4 2 0
2-l 250-l 2-kl
0 2 4 6 8 10 12 14 16 18 Run time (days)
Online pH
VCC (1e5 cell ml–1) 160
120
80
40
0 0 2 4 6 8 10 12 14 16 18
Run time (days)
Lactate (g l–1) 4
3
2
1
0 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Run time (days) Run time (days)
pCO2 (mm Hg) Osmolality (mOsm)
2-l 250-l 2-kl
2-l 250-l 2-kl
%Viable 120
80
40
0 0 2 4 6 8 10 12 14 16 18
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%HMW %Monomer
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G0 G1 G2 Man5 G0-F
Figure 8 Titer, growth, metabolism, and control profiles of mAb X in 2-, 250-, and 2000-l runs.
258 Bioreactors – Design
Figure 9 Product quality of mAb X in 2-, 250-, and 2000-l runs.
Very comparable results were obtained, in terms of cell density, cell viability, metabolites, and product titer (Figure 10). Viable cell densities of 20–25 � 106 cells ml−1 were reproducibly achieved, resulting in the production of 7–8 g l−1 IgG after 22 days on all the scales. The 250-l bioreactor also showed equivalent control of the process parameters, when compared to the lower scale.
Viable cell density (×10E6 cells ml–1) 30 100%
25 80%
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0 0%
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Disposable Bioreactors 259
Figure 10 Comparison of culture growth, titer, and metabolite profiles for the SUB bioreactors at different scales compared to a 4-l stirred-tank bioreactor with PER.C6® cells.
Viable cell density (×10E6 cells ml–1) Viablility (%) 180 100 160 140 80
120 100
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10 10
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260 Bioreactors – Design
Figure 11 Comparison of culture growth, titer, and productivity in a 50-l SUB as compared to a 1-l conventional stirred-tank bioreactor applying the XD process with PER.C6 cells.
2.19.2.4.3 PER.C6® culture (XD® process) in single-use bioreactors A highly intensified cell culture process has been developed by DSM Biologics [17], termed XD®, in which both cells and product are retained in a stirred-tank bioreactor; fresh feed is continuously supplied and waste byproducts are continuously removed. Viable cell densities of over 100 � 106 cells ml−1 and product titers above 10 g l−1 have been repeatedly attained on lab scale using multiple PER.C6® and CHO clones producing a range of products (monoclonal antibodies and recombinant proteins). Recently, the XD® process has been successfully scaled up to disposable bioreactors, using an IgG-producing PER.C6® cell line. The scale-up experiments were performed in a 2-l glass Applikon bioreactor and a 50-l Hyclone SUB controlled by a DCU3 station (Sartorius). Cells and product were retained in the bioreactor by an ATF-2 and ATF-6 device (Refine Technology), respectively. Similar conditions of temperature, pH, DO, and pCO2 control, and the same perfusion strategy were used. The same power input per volume was applied to all the scales.
Similar cell densities of above 150 � 106 cells ml−1 were achieved at both scales (Figure 11), demonstrating that disposable bioreactors are suitable for supporting very high cell density cultivations of mammalian cell lines, by providing the required mass transfer. IgG productivity in the SUB also equaled the downscaled run.
2.19.3 Conclusions
SUBs have become commonly accepted by industry and academic laboratories, mainly due to their advantages including, ease of use, flexibility, and cost. There are generally two types of bioreactors available: bioreactors with pillow-shaped bags where mixing and mass transfer is induced by a wave-type motion of the fluid and the so-called SUBs, having tank liner bags equipped with disposable stirrers that more closely mimic operationally, the traditional stirred bioreactors. The primary advantage of SUBs is their flexibility and scalability compared to the standard stirred bioreactors. Results with CHO cells and PER.C6® cells show their excellent potential in cell culture, but, due to the low mass transfer capacity, the SUBs are only suited for cell culture. Reactors are available from different suppliers in a range of 50–2000 l working volume. The pillow-shaped bioreactors are mainly used at smaller scales up to 100 l. Larger volumes in these types of bags become less practical. However, Wave-type bioreactors are widely accepted and used in the biopharmaceutical industry. Cultivations in Wave-type bioreactors result in performance comparable to the standard (small scale) stirred glass or stainless steel reactors.
Disposable Bioreactors 261
The CELL-tainer® bioreactor, applying a two-dimensional rocking motion, provides the potential of using a SUB not only in intensive cell culture applications (such as perfusion or the XD process from DSM Biologics) but also in microbial, yeast, and fungal cultures due to its ability to deliver high mass transfer. This widens the application of single-use bioreactors even to the more traditional biotechnology processes.
While much of this section has focused on specific disposable bioreactor technologies that are already on the market, perhaps the key conclusion that can be drawn from this information is that there is a definitive trend toward the use of disposables. As the acceptance level and market for single-use technologies has grown, so has the number of companies offering new products. It can be anticipated that new and improved disposable technologies will continually emerge, allowing for implementation into areas that are currently beyond the reach of today’s disposable technologies.
References
[1] Kybal J and Sikyta B (1987) A device for cultivation of plant and animal cells. Biotechnology Letters 7: 467–470. [2] Singh V (1999) Disposable bioreactor for cell culture using wave-induced agitation. Cytotechnology 30: 149–158. [3] Wurm F (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology 22: 1393–1398. [4] Kadarusman J, Bhatia R, McLaughlin J, and Lin WR (2005) Growing cholesterol dependent NS0 myoloma cell line in the wave bioreactor system: Overcoming cholesterol–polymer
interaction by using pretreated polymer or inert fluorinated ethylene propylene. Biotechnology Progress 21: 1341–1346. [5] Singh V (2003) Disposable perfusion bioreactor for cell culture. US2003/0036192 A1. [6] Mikola M, Seto J, and Amanullah A (2007) Evaluation of a novel Wave Bioreactor® cellbag for aerobic yeast cultivation. Bioprocess and Biosystems Engineering 30: 231–241. [7] Eibl R and Eibl D (2006) Design and use of the Wave Bioreactor for plant cell culture. In: Dutta Gupta S and Ibaraki Y (eds.) Plant Tissue Culture Engineering. Dordrecht: Springer. [8] Namdev PK and Lio P (2000) Assessing a disposable bioreactor for attachment-dependent cell cultures. BioPharm 13: 44–50. [9] Palazón J, Eibl R, Lettenbauer C, et al. (2003) Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor. Planta Medica 69: 344–349. [10] Weber W, Geisse S, and Memmert K (2002) Optimisation of protein expression and establishment of the Wave Bioreactor® for baculovirus/insect cell culture. Cytotechnology 38:
77–85. [11] Pierce L and Shabram PW (2004) Scalability of a disposable bioreactor from 25 L–500 L in perfusion mode with a CHO-based cell line: A tech review. BioProcessing Journal 3:
July/August. [12] Tang YJ and Hamel JF (2007) Perfusion culture of hybridoma cells for hyperproduction of IgG(2a) monoclonal antibody in a wave bioreactor-perfusion culture system.
Biotechnology Progress 23(1): 255–264. [13] Van der Heiden P, Buevink, M, and Oosterhuis NMG (2007) Method and Apparatus for Cultivating Cells Utilizing Wave Motion. International Publication Number WO 2007/
001173 A2. [14] Oosterhuis NMG, Peeters MAE, and Van der Heiden P (2008) Improved Flexible Bioreactor. WO 2008/153401/A1. [15] Eibl R and Eibl D (2009) Application of disposable bag bioreactors in tissue engineering and for the production of therapeutic agents. Advances in Biochemical Engineering/
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Programme & Book of Abstracts, 21st Meeting of the ESACT. Dublin, Ireland, 7–10 June, p. 411.
Relevant Websites
http://www.applikon-bio.com – Applikon Biotechnology. http://www.atmi-lifesciences.com – ATMI; Life Sciences. http://www.cellutionbiotech.com – Cell tainer. http://www.cellexusinc.com – Cellexus. http://www4.gelifesciences.com – GE Healthcare. http://www.kuhner.com – Kuhner. http://www.sartorius-stedim.com – Sartorius Stedim Biotech. http://www.seahorsebio.com – Seahorse Bioscience. http://www.thermo.com – Thermo Scientific. http://www.xcellerex.com – Xcellerex.
