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**Chapter 5**

**Provisional chapter**

**Development, Engineering and Biological**

**Development, Engineering and Biological** 

Cedric Schirmer, Thomas Nussbaumer, Reto Schöb,

Cedric Schirmer, Thomas Nussbaumer, Reto Schöb,

a valuable complement. Finally, a case study is presented.

mass transfer coefficient, development, cultivation system

Ralf Pörtner, Regine Eibl and Dieter Eibl

Ralf Pörtner, Regine Eibl and Dieter Eibl

http://dx.doi.org/10.5772/intechopen.79444

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Characterization of Stirred Tank Bioreactors**

**Characterization of Stirred Tank Bioreactors**

DOI: 10.5772/intechopen.79444

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

**Keywords:** stirred bioreactor, characterization, mixing time, power input, volumetric

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 [1]. They are the most frequently used bioreactor systems as they are suitable for various

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

#### **Chapter 5 Provisional chapter**

#### **Development, Engineering and Biological Characterization of Stirred Tank Bioreactors Development, Engineering and Biological Characterization of Stirred Tank Bioreactors**

DOI: 10.5772/intechopen.79444

Cedric Schirmer, Thomas Nussbaumer, Reto Schöb, Ralf Pörtner, Regine Eibl and Dieter Eibl Cedric Schirmer, Thomas Nussbaumer, Reto Schöb, Ralf Pörtner, Regine Eibl and Dieter Eibl

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79444

#### **Abstract**

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.

**Keywords:** stirred bioreactor, characterization, mixing time, power input, volumetric mass transfer coefficient, development, cultivation system

#### **1. Introduction**

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 [1]. They are the most frequently used bioreactor systems as they are suitable for various

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

expression systems, currently using predominantly recombinant *Escherichia coli* (*E. coli*) strains or Chinese hamster ovary (CHO) cell lines [2–7].

**2.1. Agitation**

**2.2. Drive**

new stirred bioreactors.

(*P/V* ≈ 5–200 W·m−3/*OTR* ≈ 0.5–8 mmol O<sup>2</sup>

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

impellers are often used for this purpose. For most applications with microorganisms, however, especially in the high cell density range (≈ 100 g·L−1 dry cell weight), higher specific power

For this purpose, radial flow impellers are used. Higher energy inputs lead to an improved gas dispersion and thus to higher oxygen transfer rates [6, 7, 10, 17–24]. Zlokarnik [19] and Mirro & Voll [17] provide an overview of the impeller types frequently used, and their field of application for the cultivation of various microbial and animal cell lines. Therefore, the process properties, in particular the mixing time, volumetric mass transfer coefficient and power input in combination with the resulting shear gradient are decisive for the impeller design to be selected [25]. Depending on the application and bioreactor size, multi-stage configurations

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 [16]. 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 [26]. 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 [13]. 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 [27–29]. However, friction with insufficient lubrication may result in attrition of the material [30]. 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

inputs and oxygen transfer rates are required (*P/V* > 5 kW·m−3 / *OTR* ≈ 300–500 mmol O<sup>2</sup>

with combinations of radial and axial flow impellers are also possible.

·L−1·h−1) as well as small cooling capacities. Axial flow

http://dx.doi.org/10.5772/intechopen.79444

Development, Engineering and Biological Characterization of Stirred Tank Bioreactors

·L−1·h−1).

89

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 [8]. 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. [9] 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 *θm* of the system, the volumetric mass transfer coefficient *kL a* and the specific power input *P/V* are determined. This enables a comparison of different bioreactor types and the definition of suitable process parameters to achieve the desired product quality and quantity [12].

A new approach based on process engineering characterization is the biological characterization. This may be a standardized *E. coli* model process enabling a reveal in the performance limits of the bioreactor system.

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.
