Representation of the highest experimentally determined kLa values (n = 5) for both drive systems used with the corresponding maximum impeller speeds and Reynolds numbers of the different impellers at a P/V of 1 W·m−3.
Stirred tank bioreactors are still the predominant cultivation systems in large scale biopharmaceutical production. Today, several manufacturers provide both reusable and single-use systems, whereas the broad variety of designs and properties lead to deviations in biological performance. Although the methods for bioreactor characterization are well established, varying experimental conditions and procedures can result in significantly different outcomes. In order to guarantee a reliable comparison and evaluation of different single-use and reusable bioreactor types, standardized methods for their characterization are needed. Equally important is the biological capability of bioreactors, which must be accessed by standardized cultivation procedures of industrially relevant organisms (bacteria, yeasts as well as mammalian and animal cell cultures). In addition, the implementation of well-defined uniform procedures for biological and engineering characterization during the development phase can support a fast assessment of the suitability of a bioreactor system. Based on stirred bioreactors, we describe the aspects of the engineering characterization in order to discuss further the biological characterization as a valuable complement. Finally, a case study is presented.
- stirred bioreactor
- mixing time
- power input
- volumetric mass transfer coefficient
- cultivation system
Stirred bioreactor systems have been used on a large scale since the beginning of antibiotics and insulin production, and are indispensable in biopharmaceutical production today . They are the most frequently used bioreactor systems as they are suitable for various expression systems, currently using predominantly recombinant
Stirred bioreactors are available as reusable systems made of steel and glass or as single-use systems in different sizes. Many well-known manufacturers offer standard stainless steel systems with volumes from 2 to 1000 L, whereby larger systems with several cubic meters are also available according to customer specifications. The smaller scale glass bioreactors are used in research and process development . The single-use systems, depending on their size, are either available as flexible bags or rigid vessels. They have become increasingly established in recent years and have found their way into biopharmaceutical productions with volumes of up to 2000 L. Eibl et al.  gives an overview of the currently available single-use systems.
In addition to the economic reasons for choosing one of the many reusable or single-use systems, they have to meet the requirements of the desired fermentation process. The design and equipment of stirred bioreactors differ in terms of their performance. The efficiency of the bioreactor is described with the help of process engineering parameters [10, 11]. Therefore, the mixing time
A new approach based on process engineering characterization is the biological characterization. This may be a standardized
It will be shown that process engineering characterization in combination with biological characterization is a simple standardized approach, which is not only necessary for the evaluation of existing bioreactor types, but also makes a valuable contribution during the development phase of new systems.
2. Theoretical background
The bioreactors used for the cultivation of microorganisms, mammalian and animal cells differ from reactors in the chemical industry in their aspect ratio (
Besides the vessel geometry, the impeller is the central element of the bioreactor. The choice of the right agitator organ has a decisive influence on the success of cultivation, as it prevents local sources and sinks. It is now possible to choose from a variety of different impeller designs, while taking into account the type of microorganism, human or animal cell line to be cultivated. Shear-sensitive cell culture processes are characterized by low energy and low oxygen input (
Traditionally, the agitator is driven via a centrally mounted shaft with the aid of a motor located above or below the bioreactor. The feedthrough of the shaft into the bioreactor has to be sealed. In the simplest case, a single-acting mechanical seal reduces the escape of organisms from the bioreactor, but bears the risk of contamination . For reasons of product safety, as well as maintaining a tight containment, double mechanical seals are predominantly used. Two pairs of sliding rings are arranged one behind the other and form an intermediate space through which a barrier fluid flows. The pressurized barrier liquid, which is often sterile condensate, prevents leakage from the fermenter . Magnetic couplings offer an alternative to complex double-acting mechanical seals. The magnetic field transfers the torque from the motor through the closed bioreactor to the impeller. The risk of contamination is further decreased by contactless power transmission . In industrial applications, both free-floating and bearing-supported impellers can be found. Bearing-supported impellers are manufactured by MAVAG AG, Millipore Corporation and ZETA Holding GmbH, among others. The impeller with one part of the magnetic coupling sits on a bearing journal where the second part of the magnetic coupling is also located. The mounting is often done by means of ceramic plain bearings [27, 28, 29]. However, friction with insufficient lubrication may result in attrition of the material . The levitation technology is used, for example, by Sartorius AG and Pall AG for mixing systems. Only the impeller with one part of the magnetic coupling is located in the vessel. The magnetic field applied causes the impeller to lift off the bottom of the container. This simple type of drive does not require a bearing, and is therefore ideally suited for use in single-use systems, whereas radially acting forces are difficult to absorb [31, 32]. As shown in our case study (see Section 3.1), the levitation technology is also suitable for new stirred bioreactors.
2.3. Characterization according to parametric and experimental approaches
Due to the large number of bioreactors available and their different process engineering properties, the choice of the right system for the requirements of a desired and successful process is decisive. The process engineering characterization allows the comparison of different systems and supports process optimization and scale-up strategies by using parametric as well as experimental approaches [11, 12]. Therefore in January 2016, DECHEMA issued a recommendation with standardized methods for obtaining reliable experimental data, which can be applied to both reusable and single-use bioreactor systems .
The dimensionless Reynolds number describes the ratio of inertial to viscous forces in a flow and describes it as laminar, transient or turbulent (
Another parameter is the maximum fluid velocity (
In order to avoid sources and sinks in the bioreactor, a homogeneous distribution of all components is required. A benchmark of homogeneity is the mixing quality, which is regarded as adequate at 95% . The mixing time
One of the most important parameters is the specific power input
Oxygen supply is essential for aerobic cultivation processes. This is ensured by the use of spargers, gassing via membranes or the fluid surface [21, 39]. The oxygen transition is defined by the oxygen transfer rate (
2.4. Characterization by biological approaches
Biological characterization focuses on the evaluation and comparison of bioreactor systems with respect to their biological performance. With the help of a model organism, it should be possible to make an exact prediction of the suitability of a bioreactor system for a desired purpose with a standardized cultivation procedure . For example, two biological test procedures with respiratory yeast and mycelium-forming fungi were developed by Adler and Fiechter  and Wagner , since the physical characterization often only provides information about optimal bioreactor design conditions and information for improved scale transfer. For this reason, DECHEMA’s
3. Case study
In this case study, the methodical procedures described above are used to develop a bearing-free magnetically driven 2 L benchtop bioreactor system, which is based on Levitronix’s freely levitating impeller technology.
3.1. Bioreactor and setup
The use of a magnetic drive without bearings enables the establishment of a seal-free, contactless and magnetically mounted bottom impeller, which offers an almost unlimited speed range and a minimized risk of contamination (Figure 1).
The impeller levitating in the bioreactor at the bottom creates a constant gap, which is made possible by the passive stabilization of the stirring element by a constantly changing magnetic field [47, 48]. For design reasons, a flat end element was chosen for the bottom, into which the BPS-i30 and BPS-i100 drives from Levitronix GmbH were introduced for the investigations. A glass cylinder with a diameter of 124.5 mm and a planar lid with nozzles for probes and the possibility of adding correction agents and feed solutions was mounted on top of it. The impellers used for the BPS-i30 drive are the geometries shown in Figure 2 with diameters of 20, 30, 40 and 50 mm and, based on this, 40, 50, 60 and 74 mm for the more powerful BPS-i100 drive. The oxygen input was made possible by means of a ring sparger with holes facing upwards. The temperature was controlled by using an electric heating and a water-flow cooling finger.
3.2. Process engineering characterization
All process engineering parameters to be investigated were determined by means of design of experiments, and the experimental data were evaluated using MODDE 10.1 (Umetrics, Sweden).
3.2.1. Power input
The specific power input (non-gassed conditions) was determined with water at a constant temperature of 25°C, and a maximum working volume according to Ref. . Because of the constructive conditions of the vessel and motor geometry, the sensor method for determining the torque was not applicable. Due to the known motor constants
Additionally, the torque was determined by numerical simulations (computational fluid dynamics (CFD)). Based on the predicted fluid flow, the power inputs of the impellers were obtained from the torque acting on the impeller and the shaft. Therefore, the fluid flow inside the bioreactor equipped with the different impellers was modeled using the finite volume solver ANSYS Fluent (ANSYS Inc., Version 16.2, USA) by using the realizable k-ϵ turbulence model for water at 25°C . The vessel walls and the impeller were treated as non-slip boundaries with standard wall functions. The axial velocity at the fluid surface was set to zero. All equations were discretized using the first-order upwind scheme and the COUPLED algorithm was chosen for pressure-velocity coupling. The fluid domain was discretized by an unstructured mesh consisting of about 8×106 to 11×106 tetrahedrons.
3.2.2. Mixing time
The mixing times were examined by the decolorization method (iodometry) at maximum working volume according to . Therefore, the bioreactor was filled with water and 2 mL·L−1 iodine potassium iodide solution (potassium iodide 40 g·L−1, iodine 20 g·L−1) and 5 mL·L−1 starch solution (1% w/v) were added under agitation at a constant temperature of 25°C. After ensuring a completely homogeneous chemical solution and a quasi-stationary fluid flow pattern, 4 mL·L−1 sodium thiosulfate solution were added and the time was measured until the color change from dark blue to colorless was achieved.
3.2.3. Volumetric mass transfer coefficient
3.3. Biological characterization
Based on the results of the process engineering characterization, the process parameters for the
The second pre-culture was also incubated in a 1-L shake flask with 150 mL medium at an initial optical density at 600 nm (
For the fed-batch process, a concentrated feed with a high glucose concentration was added into the bioreactor after the initial glucose had depleted. To maintain a constant growth rate, an exponential profile was used [51, 52]. The feed medium was formulated with the following concentrations (g·L−1): glucose (655.3), MgSO4·7H2O (16.02), CaCl2·2H2O (43·10−3), ZnSO4·7H2O (15·10−3), MnSO4·H2O (85·10−3), Na2-EDTA·2H2O (85·10−3), FeCl3·6H2O (71·10−3), CuSO4·5H2O (14·10−3) and CoCl2·6H2O (15·10−3). In contrast to the batch process, the
The batch fermentations had a starting volume of 2 L, whereas the fed-batch started with 1.3 L to ensure an appropriate covering of all sensors and heating and cooling devices. After reaching an
In the run-up to the experimental investigations, the new bioreactor system with the magnetic drive was numerically examined with regard to the process engineering parameters regarding its suitability for the cultivation of microorganisms. As expected, the specific power input shows an exponential increase with rising rotational speed (Figure 3). However, it also becomes apparent that with the weaker BPS-i30 drive, only the impellers with a diameter of 20 and 30 mm are in the range of microbial requirements with
The experimentally determined specific power inputs show only minor deviations compared to the numerically determined values, whereby larger differences result in increasing rotational speed. The largest deviation for the 20 mm impeller at 7000 rpm (
With regard to the mixing time, Figure 4 shows that all impellers with a specific power input of 1 kW·m−3 and above meet a required mixing time of
|Impeller diameter [mm]||BPS-i30||BPS-i100|
The experimentally determined
Based on the process engineering investigations, the cultivation for biological characterization was carried out. The impellers 20 and 40 mm (BPS-i30/BPS-i100) used demonstrated identical behavior during the process up to hour 6 with respect to biomass, glucose and acetate concentrations as well as in the
In reusable pilot bioreactors for microbial applications with 30 and 100 L previously tested, only optical densities of 39 ± 5 at higher oxygen transport rates of 735 and 745 h−1 were obtained . This fact can only be attributed to the considerably shorter mixing times of 2.77 and 3.47 s (20 mm/40 mm) in the 2 L scale shown here. These were determined in the mentioned larger systems with 8–10 s. The additional oxygen uptake rate
The results of the fed-batch cultivations with the BPS-i100 system presented in Figure 7 show an expected higher biomass concentration with an
The determined process engineering parameters demonstrate that the newly developed bioreactor system can be used for the cultivation of shear-sensitive animal cells as well as microbial cells up to the high cell density range. In this way, the process engineering parameters for all impellers with specific power inputs of up to 500 W·m−3 show suitability for animal cell culture processes. The
In addition, due to the simple design of the system, with the elimination of seals and bearings, by using a magnetic drive with a freely levitating impeller the bioreactor is almost maintenance-free and the risk of contamination is reduced. It also facilitates cleaning and the easy and fast change of impeller types for different applications. Furthermore, the bioreactor system offers a high turn down ratio allowing an easy-to-scale process.
While conventional microbial processes can also be implemented with the less powerful BPS-i30 drive, the BPS-i100 in combination with the 40 mm impeller is recommended for high cell density microbial processes. This is demonstrated by nearly a doubling of the optical densities in the
So it comes as no surprise that the specific power inputs obtained in the case study with the new bioreactor provide comparable results to other microbial bioreactor systems described in the literature. For bioreactors with sizes of 1–100 L, these are between 2.5 and 20 kW·m−3 [42, 67, 68, 69], whereby a minimum requirement of >5 kW·m−3 is generally assumed . The achieved mixing numbers are partly
The combination of DECHEMA’s recommendation for process engineering characterization and the
The investigated process engineering parameters allow the estimation of its optimal working areas and limits. In addition, it allows a selection of a suitable impeller design to increase the productivity of biopharmaceutical processes. The impeller with a diameter of 40 mm in combination with the more powerful BPS-i100 drive shows the highest
|CCOS||culture collection of Switzerland|
|CFD||computational fluid dynamics|
|CHO||Chinese hamster ovary cell line|
|CHO XM 111–10||SEAP secreting cell line|
|E. coli||Escherichia coli|
|SEAP||secreted alkaline phosphatase of the placenta|
|CO2||present oxygen concentration [mmol·L−1]|
|CO2∗||maximum oxygen concentration [mmol·L−1]|
|μ||specific growth rate [h−1]|
|a||phase boundary interface [m−1]|
|cH||mixing number [−]|
|d||impeller diameter [m]|
|D||vessel diameter [m]|
|DCW||dry cell weight [g·L−1]|
|DO||dissolved oxygen [%]|
|H||vessel height [m]|
|H/D||ratio of vessel height to diameter [−]|
|kL||mass transfer coefficient [m·h−1]|
|kLa||volumetric mass transfer coefficient [h−1]|
|Kt||motor torque constant [N·m·A−1]|
|Md||dead weight torque [N·m]|
|N||number of impeller revolutions [rps]|
|OD600||optical density at 600 nm [−]|
|OTR||oxygen transfer rate [mmol·L−1·h−1]|
|OUR||oxygen uptake rate [mmol·L−1·h−1]|
|P/V||specific power input [W·m−3]|
|R2||regression coefficient [−]|
|Re||Reynolds number [−]|
|utip||tip speed [m·s−1]|
|β||gassing rate [vvm]|
|θm||mixing time [s]|