Search

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult. This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 < Re < 2·104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 ≈ 0.3 and P0 ≈ 4.5 for the maximum Reynolds numbers in the different bioreactors.

Power input is an important engineering and scale-up/down criterion in stirred bioreactors. However, reliably measuring power input in laboratory-scale systems is still challenging. Even though torque measurements have proven to be suitable in pilot scale systems, sensor accuracy, resolution, and errors from relatively high levels of friction inside bearings can become limiting factors at smaller scales. An experimental setup for power input measurements was developed in this study by focusing on stainless steel and single-use bioreactors in the single-digit volume range. The friction losses inside the air bearings were effectively reduced to less than 0.5% of the measurement range of the torque meter. A comparison of dimensionless power numbers determined for a reference Rushton turbine stirrer (NP = 4.17 ± 0.14 for fully turbulent conditions) revealed good agreement with literature data. Hence, the power numbers of several reusable and single-use bioreactors could be determined over a wide range of Reynolds numbers between 100 and >104. Power numbers of between 0.3 and 4.5 (for Re = 104) were determined for the different systems. The rigid plastic vessels showed similar power characteristics to their reusable counterparts. Thus, it was demonstrated that the torque-based technique can be used to reliably measure power input in stirred reusable and single-use bioreactors at the laboratory scale.

This application note describes the cultivation of Chinese hamster ovary (CHO) suspension cells in the Finesse SmartGlass vessel bioreactor with a maximum working volume of 2.0 L. Using chemically defined minimal media, cell densities of up to 7.44 × 106 cells/mL were achieved. Recombinant secreted embryonic alkaline phosphatase (SEAP) expression was induced by medium exchange and temperature shift. Maximum SEAP activities of up to 63 U/mL were reached. A novel stirred glass bioreactor suitable for cell culture applications at benchtop scale was designed by Finesse Solutions, Inc. With a maximum working volume of 2.0 L, this bioreactor is controlled by the Finesse G3Lab controller.

During the past 10 years, single-use bioreactors have been well accepted in modern biopharmaceutical production processes targeting high-value products. Up to now, such processes have mainly been small- or medium-scale mammalian cell culture-based seed inoculum, vaccine or antibody productions. However, recently first attempts have been made to modify existing single-use bioreactors for the cultivation of plant cells and tissue cultures, and microorganisms. This has even led to the development of new single-use bioreactor types. Moreover, due to safety issues it has become clear that single-use bioreactors are the “must have” for expanding human stem cells delivering cell therapeutics, the biopharmaceuticals of the next generation. So it comes as no surprise that numerous different dynamic single-use bioreactor types, which are suitable for a wide range of applications, already dominate the market today. Bioreactor working principles, main applications, and bioengineering data are presented in this review, based on a current overview of greater than milliliter-scale, commercially available, dynamic single-use bioreactors. The focus is on stirred versions, which are omnipresent in R&D and manufacturing, and in particular Sartorius Stedim’s BIOSTAT family. Finally, we examine development trends for single-use bioreactors, after discussing proven approaches for fast scaling-up processes.

Suspension cultures, in which human mesenchymal stem cells (hMSCs) are cultivated on microcarriers in scalable single-use stirred bioreactor types, have been shown to be a promising alternative to planar flask cultures. However, stirred single-use bioreactors were originally developed for production processes with robust, permanent cell lines. hMSCs are adherent primary cells and thus expanding them in such bioreactor systems imposes more stringent requirements on bioreactor systems. For low-serum conditions (5%) and different types of stirred single-use bioreactors, a suspension criteria-based approach for expanding human adipose tissue-derived mesenchymal stem cells (hASCs) from mL- to pilot scale was successfully developed. For process scale-up, experimental and numerical investigations were performed to (1) predict optimum impeller speeds, (2) determine the main engineering parameters (local shear stress, turbulent dissipation rate, Kolmogorov microscale), and (3) verify suspension criteria NS1 and NS1u for rapid process transfer from 100 mL to 2 L and 35 L cultures. Using optimized mediummicrocarrier combinations as well as NS1 and NS1u as scale-up factors, total hASC quantities between 3 •107 (100 mL scale) and 1 • 1010 (35 L scale) were obtained. The cell quantities obtained are the highest reported to date for scalable single-use bioreactors under low-serum conditions.

Mechanical stress caused to transfected Chinese hamster ovary (CHO) suspension cells by reusable and single-use magnetically levitated, bearingless centrifugal pumps was investigated. Cell death rates were determined for different pump speeds and compared with data from a peristaltic and a 4-piston diaphragm pump. Furthermore, the fluid distribution inside the PuraLev® 200 pump was modeled using computational fluid dynamics. The results reveal considerably lower mechanical stress to CHO cells caused by the magnetically levitated bearingless centrifugal pumps than by the peristaltic and diaphragm pump.

Since design, construction and evaluation of bioreactors for large-scale production is costly and time consuming, computational methods may give some insights into the fluid mechanics within bioreactors. Thus, critical limiting factors, such as insufficient mixing as well as inhomogeneous nutrient and oxygen mass transfer, may be identified early in the design process. Although advanced experimental techniques such as laser Doppler anemometry and particle image velocimetry are also reliable, they are too time consuming to characterize the complete flow pattern in industrial scales and rely on optical accessibility. Therefore, the knowledge of flow characteristics provided by computational fluid dynamics (CFD) is indispensable for the rational design of bioreactors. Based on previously published reviews, the present work summarizes the latest publications on the usage of CFD to characterize and scale-up bioreactors used in biotechnological processes. Selected models that are used to predict the fluid flow pattern and key engineering parameters of commonly used bioreactors are described. Related issues, such as grid dependency of CFD results and the requirement for experimental verification are also addressed. Finally, an overview of proposed but not yet feasible CFD applications is presented, including fluid–structure interaction, the use of direct numerical simulation and the coupling fluid flow and chemical reactions.