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.

Single-use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single-use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein-free cell culture medium that had been used to extract leachables from freshly gamma-irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma-irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma-irradiation, have been established to ensure lot-to-lot consistency. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1171–1176, 2014

An in 2016 published DECHEMA guideline concerning process engineering characterization and a new Escherichia coli model process were utilized for the qualification of two geometrically similar stirred stainless steel bioreactors (30 L and 100 L working volume). The achieved results demonstrate that performing an additional biological model process is a valuable complement to the process engineering characterization. Optical densities of 27 (100 L) and 39 (30 L) were reached in the batch cultivation process.

During the last decades single‐use bioreactors have become widely accepted in the biopharmaceutical industry. Single‐use technologies bring many advantages over conventional solutions, such as a reduced investment and operational cost as well as an optimized time‐to‐market. So far, this type of bioreactor is mainly used for cell culture applications. Results for microbial fermentations are rarely found, commonly due to limited oxygen transfer rates. The aim of this study was to establish a high cell density fermentation process for a recombinant Escherichia coli considering a rocking motion type and an adapted stirred 50‐L single‐use bioreactor. By using the design space approach an OTRmax/OURmax model was established. The feeding strategies were optimized and verified based on a model for both single‐use systems to achieve high cell densities. In both single‐use bioreactors a recombinant protein was successfully expressed.

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Single‐use bioreactors continue to gain large interest in the biopharmaceutical industry. They are frequently used for mammalian cell cultivations, e.g. production of monoclonal antibodies and vaccines. This is motivated by several advantages of these bioreactors such as reduced risk of cross contaminations or short lead times. Single‐use bioreactors differ in terms of shape, agitation principle, and gassing strategy. Hence, a direct process transfer or scale‐up can be a challenge. Conventional stirred tank bioreactor designs are therefore still considered as the gold standard due to their well‐defined and characterized properties. Based on this knowledge, a stirred single‐use bioreactor family from 50 to 2000 L scale was developed with geometrical ratios similar to conventional reusable systems. To follow a quality by designapproach, the single‐use bioreactor family evaluated here was characterized by using process engineering methods such as the power input per volume, the mixing time, and the kLa value. The process engineering characterization demonstrates that these systems are suitable for cultivations of mammalian cells, even for high cell density and high titer applications. Based on the data, a scale‐up or process transfer is possible between this bioreactor family as well as reusable systems. Therefore, this bioreactor family represents a major progress for the single‐use technology.

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: Microbial fermentations are of major importance in the field of biotechnology. The range of applications is rather extensive, for example, the production of vaccines, recombinant proteins, and plasmids. During the past decades single-use bioreactors have become widely accepted in the biopharmaceutical industry. This acceptance is due to the several advantages these bioreactors offer, such as reduced operational and investment costs. Although this technology is attractive for microbial applications, its usage is rarely found. The main limitations are a relatively low oxygen transfer rate and cooling capacity. The aim of this study was to examine a stirred single-use bioreactor for its microbial suitability. Therefore, the important process engineering parameters volumetric mass transfer coefficient (k L a), mixing time, and the heat transfer coefficient were determined. Based on the k L a characteristics a mathematical model was established that was used with the other process engineering parameters to create a control space. For a further verification of the control space for microbial suitability, Escherichia coli and Pichia pastoris high cell density fermentations were carried out. The achieved cell density for the E. coli fermentation was OD600 = 175 (DCW = 60.8 g/L). For the P. pastoris cultivation a wet cell weight of 381 g/L was reached. The achieved cell densities were comparable to fermentations in stainless steel bioreactors. Furthermore, the expression of recombinant proteins with titers up to 9 g/L was guaranteed.