Pharmaceutical Review
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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 optimiz ations 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],