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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.

During the past five years, the number of single-use bioreactors used in biopharmaceutical research and production has increased tremendously. This increase has been particularly associated with mammalian cell culture processes from small- to medium-scale volumes. Even though nowadays customers can choose from a multitude of 2nd and 3rd generation single-use bioreactors, ranging from mL- up to m3-scale, there is a lack of knowledge of their engineering parameters. Different approaches have been applied to characterization investigations, resulting in an inability to compare different single-use bioreactors with each other and their reusable counterparts, creating an obstacle to a systematic approach to scaling-up the process. This article describes parametric, experimental and computer-based numeric methods for biochemical engineering characterization of single-use bioreactors, which have already been used successfully for the characterization of their reusable counterparts. For the first time, these methods have been evaluated in terms of their practical application.

The innovative travelling wave bioreactor (TWB), an orbitally shaken system, developed specifically for cell culture applications, is investigated. In this study, engineering parameters covering power input, mixing time and oxygen mass transfer obtained in two TWB prototypes are presented for the first time. Based on the results, cultivation parameters for CHO suspension cell cultivations were defined. Cell growth and substrate consumption were comparable with a wave-mixed reference bioreactor, which demonstrates the bioreactor's applicability for cultivations of mammalian cell cultures for the first time.

In the travelling wave bioreactor (TWB) an orbital movement is used to set up a quasi-periodic wave motion in the partially filled, toroidal shaped vessel. Based on computational fluid dynamics (CFD) models, this study presents detailed analyses of the fluid flow in the TWB for a wide range of operational conditions. Even though the wave propagation is shown to be greatly influenced by the rotation, it was found that the regular vessel shape facilitates a predominantly tangential flow with low back mixing, irrespective of the operational conditions. Hence, the influence of different bioreactor geometries without and with baffles is examined. The study indicates that CFD models can be used as a valuable tool for bioreactor development, because it provides a deep insight into the bioreactor's hydrodynamics and can help to reduce the number of prototypes.

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.

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping process shear forces occur which can lead to qualitative and/or quantitative product loss. To calculate the mechanical stress with limited experimental expense, an oil-water emulsion system was used, whose suitability was demonstrated for drop size detections in bioreactors. As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes need to be counted over the experimental time of shear stress investigations. In previous studies, the inline endoscopy has been shown to be an accurate and reliable measurement technique for drop size detections in liquid/liquid dispersions. The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. In order to express the drop size, the Sauter mean diameter d32 was used as the representative diameter of drops in the oil-water emulsion. The results showed low variation in the Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating the reliability of the measurement technique.

The potential of human mesenchymal stem cells (hMSCs) for allogeneic cell therapies has created a large amount of interest. However, this presupposes the availability of efficient scale-up procedures. Promising results have been reported for stirred bioreactors that operate with microcarriers. Recent publications focusing on microcarrier-based stirred bioreactors have demonstrated the successful use of Computational FluidDynamics (CFD) and suspension criteria (NS1u,NS1) for rapidly scaling up hMSC expansions from mL- to pilot scale. Nevertheless, one obstacle may be the formation of large microcarrier-cell-aggregates, which may result in mass transfer limitations and inhomogeneous distributions of stem cells in the culture broth.The dependence of microcarrier-cell-aggregate formation on impeller speed and shear stress levels was investigated for human adipose derived stromal/stem cells (hASCs) at the spinner scale by recording the Sauter mean diameter (d 32) versus time. Cultivation at the suspension criteria provided d 32 values between 0.2 and 0.7 mm, the highest cell densities (1.25 × 106 cells mL−1 hASCs), and the highest expansion factors (117.0 ± 4.7 on day 7), while maintaining the expression of specific surface markers. Furthermore, suitability of the suspension criterion NS1u was investigated for scaling up microcarrier-based processes in wave-mixed bioreactors for the first time.

A two-step process for the fractionation of the proteins in native whey was developed at lab scale. An anion- and cation-exchanger membrane device was used. The process was transferred from lab to pilot scale in order to demonstrate the industrial applicability. Various process parameters as well as adsorption and mass transfer phenomena were investigated in order to gain deeper process understanding. Based on these studies the most efficient process in terms of productivity and fraction purity was proposed., Es wurde ein zweistufiges Verfahren im Labormaßstab entwickelt, anhand dessen das Proteingemisch in nativer Molke in seine Einzelkomponenten aufgetrennt werden konnte. Dazu wurden Anionen- und Kationentauscher auf Membranbasis verwendet. Der Prozess wurde auf Technikumsmaßstab hochskaliert, um eine industrielle Anwendbarkeit zu demonstrieren. Es wurden verschiedene Prozessbedingungen sowie Adsorptions- und Stofftransportphänomene untersucht, um basierend auf einem vertieften Prozessverständnis den optimalen Prozess hinsichtlich Produktivität und Proteinreinheit abzuleiten.

A two-step process for the fractionation of the proteins in native whey was developed at lab scale. An anion- and cation-exchanger membrane device was used. The process was transferred from lab to pilot scale in order to demonstrate the industrial applicability. Various process parameters as well as adsorption and mass transfer phenomena were investigated in order to gain deeper process understanding. Based on these studies the most efficient process in terms of productivity and fraction purity was proposed., Es wurde ein zweistufiges Verfahren im Labormaßstab entwickelt, anhand dessen das Proteingemisch in nativer Molke in seine Einzelkomponenten aufgetrennt werden konnte. Dazu wurden Anionen- und Kationentauscher auf Membranbasis verwendet. Der Prozess wurde auf Technikumsmaßstab hochskaliert, um eine industrielle Anwendbarkeit zu demonstrieren. Es wurden verschiedene Prozessbedingungen sowie Adsorptions- und Stofftransportphänomene untersucht, um basierend auf einem vertieften Prozessverständnis den optimalen Prozess hinsichtlich Produktivität und Proteinreinheit abzuleiten.