Regular articleCharacterization and comparison of ATF and TFF in stirred bioreactors for continuous mammalian cell culture processes
Graphical abstract
Introduction
The ongoing improvement of equipment and processes for the production of recombinant proteins has been fueled by the ever growing market of biopharmaceuticals [1]. Although well-established fed-batch processes are currently still favored in industrial production, perfusion cultures are a valuable alternative for unstable proteins and special product quality requirements. Moreover, continuous cultivation has regained interest when used for the inoculation of high cell density fed batch cultures [2], [3], [4], [5] or cell banking [6], [7], [8]. More recently, the first direct connection of a perfusion bioreactor to a chromatographic protein capture step has been reported [9]. The integration of both upstream production and downstream purification is beneficial, offering cost advantage due to smaller equipment size at higher volumetric productivity. In addition, the short residence time of the desired protein in the bioreactor, combined with the steady state operation favors a constant and possibly improved product quality [10], [11].
The incorporation of a cell retention device is a necessity to achieve higher viable cell densities, which significantly increases nutrient consumption compared to classical fed-batch mode [12]. The higher metabolic demands are accompanied with elevated requirements in terms of oxygen mass-transfer, CO2 removal and homogenization of the culture broth. Spatial gradients in pH, nutrients and gaseous composition, particularly in the cell retention device, may harm attainable cellular growth and productivity. Therefore, the knowledge of bioreactor hydrodynamics, e.g., gas–liquid mass transfer and mixing efficiency, aid the successful design of the reactor system and the definition of suitable operating parameters. In the pursuit of an integrated process, the application of external cross flow filtration employing hollow fiber modules is the method of choice, offering the direct harvest of cell free supernatant. Other technologies based on sedimentation [3], [13], [14], [15], [16], centrifugation [17], [18] or ultrasound [19], [20], [21] are limited to only partial retention of cells and require a further processing step to exclude the residual biomass. In general, two different flow modes in operation of the cross-flow filtration device can be distinguished, the first being tangential flow (TFF) and the second being alternating tangential flow (ATF).
TFF systems proved to be suitable for the perfusion culture of mammalian cells [8], [22], [23], [24]. Although filter utilization can be limited as a result of membrane fouling, the application of TFF devices for biological material is well characterized and scale-up parameters have been identified [25], [26], [27]. The reported impact on cell integrity from the pump driving the culture broth and the laminar shear field in the fiber lumen [28] make the determination of suitable operating ranges to prevent cell damage necessary [29], [30], [31].
More recently, the commercially available ATF device (Repligen, USA) has gained increasing attention due to its superior performance. The ATF system has been applied for high cell density seeding [2], [32], cryopreservation [7], [33] and the integrated continuous production of biotherapeutics [9]. A diaphragm pump periodically cycles the cell culture broth through a hollow fiber module, thereby creating an alternating flow. The application of a diaphragm pump to drive the external loop instead of commonly used peristaltic pumps reduces the exposure of cells to high hydrodynamic stress values. Long term viable cell cultures have been shown for CHO cells, achieving cell densities of up to 130 × 106 cells/mL [8]. More importantly, the periodic pressure and exhaust cycle in the ATF creates a backflush through the filter pores from the permeate side, changing the hydrodynamics of the system significantly. The induced change in flow direction and the resulting pressure gradient on the fiber inner surface is thought to mitigate membrane fouling [34]. Besides a recent study showing the dependence of membrane fouling to cell and antifoam concentration at varying permeate flow rates [35], data on the physical characterization of the ATF is limited.
Comparable growth and productivity of CHO cells in a wave bioreactor setup with either ATF or TFF has been shown [8], [33]. However, higher cell densities (>130 × 106 cells/mL) in the ATF were limited by the failure to move the viscous cell culture broth. Both setups showed significant retention of the produced antibody in the hollow fiber module due to filter fouling.
In this study two identical stirred tank perfusion bioreactor setups are compared, the first employing the commercial ATF and the second being equipped with a bearingless centrifugal pump, running in tangential flow mode for cell retention. Hydrodynamic characterization in terms of shear stress exposure and oxygen mass transfer as well as mixing efficiency is carried out to define physiological and safe operating ranges. The validity of this approach is confirmed by the cultivation of mammalian cells in perfusion mode during three weeks. The obtained results clearly illustrate the importance of physical bioreactor characterization to provide optimal process conditions, in particular regarding high cell density processes.
Section snippets
Bioreactor description
Two perfusion setups equipped with one of the two retention systems considered in this work, TFF and ATF, are shown in Fig. 1. The setup is based on a 2.5 L DASGIP® benchtop bioreactor system (Eppendorf AG, Switzerland) with 13 cm tank diameter (T). In case of the TFF system, the glass vessel has been modified with a bottom outlet to allow the direct attachment of the bearingless centrifugal pump (PuraLev® 200MU, Levitronix AG, Switzerland) to circulate the culture broth through the external
Characterization of the reactor setups
Given the increased cell densities in perfusion mode, the requirements for higher gas–liquid mass transfer (e.g., oxygen supply, CO2 removal) and mixing are evident. Their understanding is crucial for the development of scalable processes. In particular, the enhanced system complexity due to a cell retention device makes a thorough system characterization necessary. Thereby, suitable operating parameters can be defined in order to maintain a viable and stable perfusion cell culture. The ATF and
Conclusion
Two small scale bioreactor systems employing a different device for cell retention were designed, aiming at a stable mammalian cell perfusion culture. In particular, a commercially available ATF system and a self-built TFF system employing a bearingless centrifugal pump were considered. A complete hydrodynamic characterization of both setups, testing two different hollow fiber modules, revealed a lower maximum shear stress in the ATF. Based on the characterization, suitable operating parameters
Acknowledgement
We would like to acknowledge Merck-Serono SA for providing the cell line and nutrition media as well as Levitronix GmbH for the supply of centrifugal pumps. This work was financially supported by the SNF Grant 206021_150744/1. MS was partially supported by a specific University Research grant of UCT (number 20/2015).
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