Stirred Tank Reactors

General

Stirred tank bioreactors (STR) are the most widely used bioreactor type to cultivate suspension cells, mainly due to the broad experience obtained in microbial

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Perfusion Stirred Tank Reactor

Rocking Motion

Figure 1 Schematic representation of the Wave™ bioreactor with wave-induced agitation. [Reprinted from Ref. (32), Copyright # 1999 with kind permission from Kluwer Academic Publishers.]

Rocking Motion

Base

Figure 1 Schematic representation of the Wave™ bioreactor with wave-induced agitation. [Reprinted from Ref. (32), Copyright # 1999 with kind permission from Kluwer Academic Publishers.]

fermentation. They have successfully been used for the cultivation of a wide variety of suspension cells and cells adapted to growth in suspension such as hybridoma cells, CHO, BHK 21, HEK293, and others. Commercial applications are the production of monoclonal antibodies (33,34), recombinant proteins such as blood coagulation factor VIII, tPA, erythropoietin, and other proteins for replacement therapy (35,36), vaccines (37), growth factors, and interferon (5). Novel applications are in gene and cell therapy such as the expansion of hematopoietic cells (14) for reconstituting in vivo hematopoiesis in patients who have undergone intensive chemotherapy, the cultivation of human T-cells for immunotherapy (15), or for the production of virus vectors (17).

A schematic drawing of a typical bioreactor setup is given in Fig. 2. Major advantages are the relative ease of handling and the familiarity of technical personnel with this reactor type. Scale-up principles are better characterized compared to other bioreactor types and mechanical design principles for sterilization-in-place (SIP) and cleaning-in-place (CIP) are known to manufacturing engineers. Furthermore, regulatory agencies are experienced with products obtained from this bioreactor type in batch, fed-batch, and perfusion modes. Especially for contract manufacturers and pilot production facilities in the pharmaceutical industry the high flexibility in terms of applicable working volumes, suitability for different cell types, operation modes, and products is a clear economic advantage.

Large-scale stirred tanks are used widely in the chemical and biochemical industries up to scales of several thousand cubic meters. For animal cells maximum working volumes of 15000 L have been reported (5,38). In the past some pharmaceutical companies have used their own engineering departments for the construction of large bioreactors. In addition, a number of manufacturers provide stirred tank reactors from 0.5 L to 15 m3. During the last 15 years, a consolidation of the number of

Structure Stirred Tank Reactors

Figure 2 Schematic drawing of stirred tank bioreactor equipped with silicone tubing for bubble free oxygen transfer. 1 reactor vessel, 2 jacket, 3 jacket connections, 4 ports for pH, temperature, pO2 electrodes, 5 sample valve with steam connection, 6 harvest valve with steam connection, 7 inoculum valve array with steam connection, 8 connection for acid, base, anti-foam, 9 three-blade segment impeller, 10 air inlet filter of aeration basket, 11 air outlet filter of aeration basket, 12 aeration basket, 13 exhaust cooler, 14 high foam alarm, 15 exhaust filter, 16 relief valve, 17 mechanical seal, 18 motor, 19 sensor port, 20 sight glass with light (not shown), 21 lateral sight glass. (Courtesy of B. Braun Biotech International.)

Figure 2 Schematic drawing of stirred tank bioreactor equipped with silicone tubing for bubble free oxygen transfer. 1 reactor vessel, 2 jacket, 3 jacket connections, 4 ports for pH, temperature, pO2 electrodes, 5 sample valve with steam connection, 6 harvest valve with steam connection, 7 inoculum valve array with steam connection, 8 connection for acid, base, anti-foam, 9 three-blade segment impeller, 10 air inlet filter of aeration basket, 11 air outlet filter of aeration basket, 12 aeration basket, 13 exhaust cooler, 14 high foam alarm, 15 exhaust filter, 16 relief valve, 17 mechanical seal, 18 motor, 19 sensor port, 20 sight glass with light (not shown), 21 lateral sight glass. (Courtesy of B. Braun Biotech International.)

Table 1 International Suppliers of Stirred Tank Bioreactors

Name Web address

Abec http://www.abec.com

Applikon http://www.applikon.com

B. Braun Biotech International http://www.bbraunbiotech.com

Bioengineering http://www.bioengineering.ch

Infors http://www.infors.ch

New Brunswick Scientific http://www.nbsc.com

bioreactor manufacturers has occurred. Major suppliers internationally active in this market segment at present are listed in Table 1.

General design criteria are derived from STRs for microbial culture (39,40) but modified to meet the requirements of the more sensitive animal cells. Specifically, the shear sensitivity of animal cells needs consideration regarding the design of impellers, use of baffles, aspect ratio, and oxygenation method.

Mixing

Agitation in stirred tanks aims for homogeneous suspension of the cells and the prevention of chemical (nutrients, waste products), physical (pH, oxygen, carbon dioxide), and thermal gradients in the vessel. Typically, large impellers (impeller diameter >0.5 x vessel diameter) with an axial fluid flow characteristics such as marine-type impellers, large paddle impellers, or segmented impellers (see Fig. 3) are used to achieve nonturbulent bulk flow patterns at minimum shear rates (41-45). Turbine impellers as used in microbial bioreactors cause damage to many cell lines and sufficient mixing requires rather high stirrer speed. Pumping capacity can be maximized and mixing times minimized by the use of large agitators operated at low stirrer rates rather than small impellers at high stirrer rates (46). As the reactor size increases, especially with increasing height, it becomes necessary to install multiple impellers to prevent compartmentalization of the fluid. In such setups attention has to be paid to optimum positioning of impellers regarding mixing times and homogeneity at a

Vibromixer
Figure 3 Schematic drawing (right). [Reprinted from Ref. Academic Publishers.]

of large paddle impeller (left) and three-blade segment impeller (42), Copyright # 1993, with kind permission from Kluwer given stirrer speed. Details regarding impeller choice, scale-up, and transitions between flow regimes are given in Ref. (47). Many other impeller types have been suggested in the past. Anchor mixers (41) provide superior mass transfer when using an aeration basket with silicon tubing wrapped around (48) due to its radial transport characteristics. Feder and Tolbert suggested a sail impeller that should provide low shear homogeneous mixing (49). Comparative studies using a shear-sensitive inorganic test system revealed, however, that lowest shear forces as determined by disruption of the flocks were obtained with a three-blade segment impeller followed by a large pitch bladed impeller (42). Different helical impeller types have been applied for insect and plant cell cultures (50,51). In combination with special baffles at the gas-liquid interface surface oxygen transfer rates of 4 up to 45 h^1 were reported for water and agar suspensions (51). The cell lift impeller (52) is characterized by a very specific design for gentle mixing by pumping the fluid upwards through an axial cylinder and gas transfer in a surrounding aeration cavity covered by a mesh screen (see Fig. 10). The annular cage impeller (53) is very similar in design but provides improved oxygen transfer. Even other mixer types such as the Vibro-mixer (54-56) and the vibrating mixer described by Monahan (57) are combined mixing and oxygen transfer devices. Blasey (56) reported on the advantageous application of the Vibromixer for keeping BHK21 cells in single cell suspension. A special tumbling membrane stirrer developed by Lehmann (1-100 L bioreactor scale) (5860) provides gentle mixing and efficient gas transfer through hydrophobic polypropylene membranes. Despite the variety of impeller types described, marine impeller designs generating an upward fluid flow with a rather low radial dispersion are dominant for suspension culture in industrial bioreactor setups.

In the literature, a number of scale-up criteria valid for agitation rate and tolerable shear forces are discussed such as impeller tip speed (61), integrated shear factor (62), or Kolmogorov's theory of isotropic turbulence (63-65). For hybridoma cells a maximum tolerable tip speed of 1 m/sec is reported for laboratory scale (1-10 L) STRs (42,66). Other authors state tolerable tip speeds of up to 2m/sec (46,67). A very important consideration for cell damage by agitation is vortex formation with bubble entrainment since this phenomenon is very likely to cause cell death (66). Other authors have investigated the effect of agitation rate on cell growth and product formation with and without sparging (68). They found that increasing stirrer speed alone did not cause cell damage but in combination with direct sparging a decrease in cell growth rate and maximum density was observed. The use of baffles is avoided in animal cell culture to minimize the generation of shear forces. If the agitation rate is too low dead zones are formed causing cell sedimentation and subsequent irreversible aggregation.

Aeration

Typical aspect ratios (height-diameter) for stirred tank reactors vary between 1:1 and 3:1. At low aspect ratios gas transfer via the headspace of the vessel is improved due to the relatively high surface to volume ratio. Stirred tank reactors used for microcarrier culture are frequently designed accordingly. However, higher aspect ratios offer advantages when direct sparging is used for oxygenation of the cell culture. Better dispersion and longer residence times of gas bubbles in the culture liquid are obtained resulting in improved gas transfer rates. Direct sparging of air or oxygen is performed with ringspargers (drilled holes > 0.5 mm diameter) similarly designed as those used in microbial fermenters or microspargers made of sintered stainless steel material (10-100 mM pore size). Especially in small bioreactors excessive sparging leads to considerable cell damage and death as the cells are disrupted when the bubbles burst at the gas-liquid interface. In larger bioreactors excessive sparging and foam formation can be minimized effectively by applying microspar-ging with pure oxygen at low flow rates with optimized pO2 control parameters. The observation that shear stress through sparging is decreased as the reactor size increases is confirmed by Henzler's (48) theoretical treatment of sparging in different reactor scales.

The gas-liquid interfacial phenomena and their effects on growth, viability, and productivity of animal cells have been extensively studied by many authors (15,69-75). For a recent review see Ref. (76). Cell damage through bursting bubbles is even increased in serum-free or protein-free media due to the missing shear protection provided by serum or albumin. Therefore, surface active substances such as Pluronic F68, Polyvinylalcohol, Methocel, and other polymers are typically added to cell culture media (77,78). Furthermore, cells might be entrapped in stable foam layers generated by excessive aeration with microspargers (79). Foaming can be reduced by the addition of antifoam agents, although these agents may negatively interfere during downstream processing. Alternatively, the use of a hydrophobic net made of polysiloxane placed onto the liquid has been proposed (80). Despite the shear and foam related challenges, direct sparging is most frequently used in stirred tank reactors larger than 10 L due to the high oxygen transfer rates provided (48,81) and the simple scale-up, handling, in situ sterilization (SIP) and cleaning-in-place (CIP) of the equipment.

Another problem encountered with microsparging is carbon dioxide (CO2) accumulation as a result of the low flow rates of pure oxygen (<0.01 vvm) applied to minimize cell disruption and foam formation. Elevated pCO2 levels decrease the medium pH and may adversely affect productivity (82,83), cell growth, or glycosyla-tion of protein products (84). Therefore, oxygen transfer and CO2 stripping needs to be balanced carefully via optimized bubble size and airflow rate. Another possibility to remove excessive CO2 is through intense ventilation of the headspace with air. Stripping can further be improved through a radial impeller directly below the liquid surface (48). Addition of HEPES (zwitterionic organic buffer) to increase the buffer capacity or sodium hydroxide or bicarbonate as corrective agents will, however, rather increase the pCO2 and osmolarity (85) and may even lead to cell damage due to high local pH values (86). To overcome these problems an optimized direct sparging strategy taking into account the CO2 accumulation was recently proposed by a research group at Bayer (87,88).

Bubble-free oxygen transfer methods were suggested for stirred tanks via thin, gas permeable silicone tubes (89,90) or microporous membranes made of PTFE

(91.92) or polypropylene (59). These systems have significantly lower oxygen transfer capacities when compared to direct sparging. Scale-up of membrane aeration devices is very limited and the largest vessels constructed provide 100-300 L working volume

Arranging many meters of tubing to maximize mass transfer and mixing is an engineering challenge. In particular, sedimentation of cells, cell aggregates, or microcarriers may occur. A comparison of oxygen transfer rates reported in the literature is given in Table 2. Oxygen consumption rates determined for different cell lines (CHO, BHK, hybridoma cells, insect cells, hematopoietic cells) are in the range of 0.02-0.6 mmol/106cells/hr (14,94-96). Based on these data it can be calculated that an oxygen transfer coefficient kIa of 0.4-4 h^1 is necessary to supply sufficient

Table 2 Comparison of Oxygen Transfer Rates kia for Different Aeration Systems Used in Stirred Tank Reactors

Aeration mode kYa (1/hr)

Parameters affecting oxygen transfer

Operation parameter range

Comment

References

Monolayer Surface

Sparging

Caged sparging Bubble free

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Responses

  • Jude
    What type of flow is in a stirrer tank reactor in mammalian?
    7 years ago
  • Gioacchino
    What is mechanical stirred tank fermenter?
    3 years ago
  • Francesca
    What is mechanical design of stirred tank reactor?
    3 years ago

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