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Bioprocess scale-up and technology transfer can be challenging due to multiple variables that need to be optimized during process development from laboratory scale to commercial manufacturing. Cell cultures are highly sensitive to key factors during process transfer across scales, including geometric variability in bioreactors, shear stress from impeller and sparging activity, and nutrient gradients that occur due to increasing blend times. To improve the scale-up and scale-down of these processes, it is important to fully characterize bioreactors to better understand the differences that will occur within the culture environment, especially the hydrodynamic profiles that will vary in vessel designs across scales. In this study, a comprehensive hydrodynamic characterization of the Ambr® 250 mammalian single-use bioreactor was performed using time-accurate computational fluid dynamics simulations conducted with M-Star computational fluid dynamics software, which employs lattice-Boltzmann techniques to solve the Navier-Stokes transport equations at a mesoscopic scale. The single-phase and two-phase fluid properties within this small-scale vessel were analyzed in the context of agitation hydrodynamics and mass transfer (both within the bulk fluid and the free surface) to effectively characterize and understand the differences that scale-down models possess when compared to their large-scale counterparts. The model results validate the use of computational fluid dynamics as an in-silico tool to characterize bioreactor hydrodynamics and additionally identify important free-surface transfer mechanics that need to be considered during the qualification of a scale-down model in the development of mammalian bioprocesses. • Multiphase hydrodynamic characterization of Ambr250 mammalian vessel using CFD. • Key scale-up parameters (mixing time, bubble size, and kLa) assessed. • Validated CFD characterization of small-scale model using experimental results. • During scale-up, free surface mass transfer is important in small-scale vessels. • CFD assessment of small-scale bioreactor helps to understand cell damage. [ABSTRACT FROM AUTHOR]

The importance of a single-use bioreactor (SUB) is continuously increasing in research and industrial fields for biopharmaceutical production. In this study, a newly developed SUB system, the CELBIC® system, applied with the unique agitation method is introduced, and its physical properties and biological applications are evaluated. The mixing time was in the range of 11.0–129.0 s, and the volumetric mass transfer coefficient, kLa, measured through surface aeration, was in the range of 2.7–15.5 h−1 at a working volume of 1–10 L. Biological evaluations using two Chinese hamster ovary (CHO) cell lines, CHO-DG44 and CHO-S cell, were carried out in the CELBIC® systems in 1 and 50 L scales, showing similar cell culture performance as that from stirred tank-type bioreactors. These results support the CELBIC® system as a new SUB system applicable to cell cultures for biopharmaceutical production. [ABSTRACT FROM AUTHOR]

Ever since agriculture started, plants have been bred to obtain better yields, better fruits, or sustainable products under uncertain biotic and abiotic conditions. However, a new way to obtain products from plant cells emerged with the development of recombinant DNA technologies. This led to the possibility of producing exogenous molecules in plants. Furthermore, plant chemodiversity has been the main source of pharmacological molecules, opening a field of plant biotechnology directed to produce high quality plant metabolites. The need for different products by the pharma, cosmetics agriculture and food industry has pushed again to develop new procedures. These include cell production in bioreactors. While plant tissue and cell culture are an established technology, beginning over a hundred years ago, plant cell cultures have shown little impact in biotechnology projects, compared to bacterial, yeasts or animal cells. In this review we address the different types of bioreactors that are currently used for plant cell production and their usage for quality biomolecule production. We make an overview of Nicotiana tabacum, Nicotiana benthamiana, Oryza sativa, Daucus carota, Vitis vinifera and Physcomitrium patens as well-established models for plant cell culture, and some species used to obtain important metabolites, with an insight into the type of bioreactor and production protocols. [ABSTRACT FROM AUTHOR]

Ever since agriculture started, plants have been bred to obtain better yields, better fruits, or sustainable products under uncertain biotic and abiotic conditions. However, a new way to obtain products from plant cells emerged with the development of recombinant DNA technologies. This led to the possibility of producing exogenous molecules in plants. Furthermore, plant chemodiversity has been the main source of pharmacological molecules, opening a field of plant biotechnology directed to produce high quality plant metabolites. The need for different products by the pharma, cosmetics agriculture and food industry has pushed again to develop new procedures. These include cell production in bioreactors. While plant tissue and cell culture are an established technology, beginning over a hundred years ago, plant cell cultures have shown little impact in biotechnology projects, compared to bacterial, yeasts or animal cells. In this review we address the different types of bioreactors that are currently used for plant cell production and their usage for quality biomolecule production. We make an overview of Nicotiana tabacum, Nicotiana benthamiana, Oryza sativa, Daucus carota, Vitis vinifera and Physcomitrium patens as well-established models for plant cell culture, and some species used to obtain important metabolites, with an insight into the type of bioreactor and production protocols.

A novel single‐use bioreactor was recently introduced to the market that is agitated by impellers suspended on flexible ropes rather than a rigid shaft. Computational fluid dynamics (CFD) models were created and validated by particle image velocimetry (PIV) to predict the bioreactor's fluid flow and mixing. The data were then used to scale‐up a Spodoptera frugiperda, subclone 9 (Sf9) insect cell‐based production of recombinant adeno‐associated virus (AAV) from a benchtop glass bioreactor to the single‐use system with 30 L working volume. This viral vector is one of the most commonly used in gene therapies. The volumetric power input was kept constant while maintaining reasonable mixing times and shear stresses between the scales. Peak cell densities of up to 7.2 × 106 cells mL−1 and maximum virus titers of 1.7 × 1011 vg mL−1 were achieved. Similar cell growth and metabolite profiles further proved the successful process transfer between the two geometrically non‐similar bioreactor systems. The pilot bioreactor yielded between 3.3 and 4.8 × 1015 vg that, depending on the therapy, can be sufficient for the treatment of a single patient. [ABSTRACT FROM AUTHOR]

During a closed connected single-use monoclonal antibody (mAb) purification process, samples for leachables screening were gathered from two parallel processes (using different capturing chromatography), from perfusion culture to final storage bags. These samples were prepared and analyzed using screening methods for HS-GC-MS, GC-MS, LC-QToF/ESI pos and neg, to be able to identify a broad spectrum of leachables. The identified compounds were sorted into sample points from different steps of the mAb process, compared with available extractables data mapped from the process equipment used. It was therefore possible to compare leachables with extractables and at the same time follow the appearance and disappearance of leachables during the process. A large number of leachables could be detected, many of which were predicted by the extractable mapping and explained by the utilized single-use equipment, for example, several ethylene glycols and laurolactam, increasing after a filtration but decreasing during the next process step. The data verifies that the purification process contains several sinks for leachables, which is a common assumption when risk-assessing extractables and leachables. The vast majority of identified leachables were removed as quickly as they were released, with the logical exception of leachables from the final storage bags plus the material and processes associated with the final step of the process., (© PDA, Inc. 2024.)

Abstract The implementation of single‐use technologies offers several major advantages, e.g. prevention of cross‐contamination, especially when spore‐forming microorganisms are present. This study investigated the application of a single‐use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040‐09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease‐inhibiting substance in the single‐use bioreactor were similar to those in the glass bioreactor.

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to manufacturing PSCs in single-use bioreactors, particularly at larger volumetric scales. This manuscript addresses these challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs. [ABSTRACT FROM AUTHOR]

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of β-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production. [ABSTRACT FROM AUTHOR]

Abstract Gene therapy and viral vaccine are becoming attractive therapeutic options for the treatment of different malignant diseases. Viral vector productions are often using static culture vessels and small volume stainless steel bioreactors (SSB). However, the yield of each vessel can be relatively low and multiple vessels often need to be operated simultaneously. This significantly increases labor intensity, production costs, contamination risks, and limits its ability to be scaled up, thus, creating challenges to meet the quantities required once the gene therapy or viral vaccine medicine goes into clinical phases or to market. Single-use bioreactor combining with microcarrier provides a good option for viral vector and vaccine production. The goal of the present studies was to develop the microcarrier bead-to-bead expansion and transfer process for HEK293T cells and Vero cells and scale-up the cultures to 50–200 l single-use bioreactors. Following microcarrier bead-to-bead transfer, the peak cell concentration of HEK293T cells reached 1.5 × 106 cells/ml in XDR-50 bioreactor, whereas Vero cells reached 3.1 × 106 cells/ml and 3.3 × 106 cells/ml in XDR-50 bioreactor and XDR-200 bioreactor, respectively. The average growth rates reached 0.61–0.68/day. The successful microcarrier-based scaleup of these two cell lines in single-use bioreactors demonstrates potential large-scale production capabilities of viral vaccine and vector for current and future vaccines and gene therapy.

The implementation of single‐use technologies offers several major advantages, e.g. prevention of cross‐contamination, especially when spore‐forming microorganisms are present. This study investigated the application of a single‐use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040‐09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease‐inhibiting substance in the single‐use bioreactor were similar to those in the glass bioreactor. [ABSTRACT FROM AUTHOR]

Herein, we described a scale‐up strategy focused on the dissolved carbon dioxide concentration (dCO2) during fed‐batch cultivation of Chinese hamster ovary cells. A fed‐batch culture process for a 2000‐L scale stainless steel (SS) bioreactor was scaled‐up from similarly shaped 200‐L scale bioreactors based on power input per unit volume (P/V). However, during the 2000‐L fed‐batch culture, the dCO2 was higher compared with the 200‐L scale bioreactor. Therefore, we developed an alternative approach by evaluating the kLa values of O2 (kLa[O2]) and CO2 [kLa(CO2)] in the SS bioreactors as a scale‐up factor for dCO2 reduction. The kLa ratios [kLa(CO2)/kLa(O2)] were different between the 200‐L and 2000‐L bioreactors under the same P/V condition. When the agitation conditions were changed, the kLa ratio of the 2000‐L scale bioreactor became similar and the P/V value become smaller compared with those of the 200‐L SS bioreactor. The dCO2 trends in fed‐batch cultures performed in 2000‐L scale bioreactors under the modified agitation conditions were similar to the control. This kLa ratio method was used for process development in single‐use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The kLa ratios for the SUBs were evaluated and conditions that provided kLa ratios similar to the 200‐L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the kLa ratio is a powerful scale‐up factor useful to control dCO2 during fed‐batch cultures. [ABSTRACT FROM AUTHOR]

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50‐L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low‐viability late fed‐batch culture, and viability was restored. The successive runs allowed optimization of the HC‐bag configuration, and cultivations with 20–25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor. [ABSTRACT FROM AUTHOR]

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to the manufacturing of PSCs in large enough quantities to meet commercial needs. This manuscript addresses the challenges for the process development of PSCs production in a bioreactor, and also presents a scalable bioreactor technology that can be a possible solution to remove the bottleneck for the large-scale manufacturing of high-quality therapeutic cells derived from PSCs. [ABSTRACT FROM AUTHOR]

Understanding the hydrodynamic conditions in bioreactors is of utmost importance for the selection of operating conditions during cell culture process development. In the present study, the two-phase flow in the lab-scale single-use bioreactor XcellerexTM XDR-10 is characterized for working volumes from 4.5 L to 10 L, impeller speeds from 40 rpm to 360 rpm, and sparging with two different microporous spargers at rates from 0.02 L min−1 to 0.5 L min−1. The numerical simulations are performed with the one-way coupled Euler–Lagrange and the Euler–Euler models. The results of the agitated liquid height, the mixing time, and the volumetric oxygen mass transfer coefficient are compared to experiments. For the unbaffled XDR-10, strong surface vortex formation is found for the maximum impeller speed. To support the selection of suitable impeller speeds for cell cultivation, the surface vortex formation, the average turbulence energy dissipation rate, the hydrodynamic stress, and the mixing time are analyzed and discussed. Surface vortex formation is observed for the maximum impeller speed. Mixing times are below 30 s across all conditions, and volumetric oxygen mass transfer coefficients of up to 22.1 h−1 are found. The XDR-10 provides hydrodynamic conditions which are well suited for the cultivation of animal cells, despite the unusual design of a single bottom-mounted impeller and an unbaffled cultivation bioreactor.

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of β-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.

• hUCM-MSC culture in Wave-Induced Motion Bioreactor was performed for the first time. • A new Cellbag design was proposed to improve cell growth. • The new Cellbag design inhibited the formation of dune-like deposits. • Expansion factors from 11.6 to 25.6 fold were achieved with the Wave-Induced Motion Bioreactor using the new Cellbag design. Mesenchymal Stromal Cells (MSCs) are considered a viable option for the treatment of several diseases in different medical areas. MSCs are present in small proportions in tissues, thus making an in vitro expansion necessary in accordance with the cGMP. For this purpose, the disposable Wave-Induced Motion Bioreactor (WIMB) is a promising alternative for MSC expansion in a safe and low shear environment. However, wave motion leads to the formation of aggregate deposits (microcarriers and cells) on the Cellbag plastic, impairing the MSC proliferation. In this study, a modification in the Cellbag design was proposed and tested in order to overcome this limitation. Human Umbilical Cord Matrix derived Mesenchymal Stromal Cells (hUCM-MSCs) were expanded in WIMB using Cultispher-S microcarriers and α-MEM medium. Two approaches were evaluated for hUCM-MSCs culture in the new Cellbag design: 1) adhesion phase in spinner and expansion in the WIMB (Expansion Factors: 5.8–8.0 fold) and 2) adhesion and expansion phases in the WIMB (Expansion Factors: 11.6–25.6 fold), both with absence of the most damaging particle deposits. These results showed that the use of WIMB with the Cellbag design modification have good potential for hUCM-MSCs expansion, while maintaining cell quality attributes. [ABSTRACT FROM AUTHOR]

Highlights • Determination of overall heat transfer coefficients in single-use bioreactors. • Comparison of determination approaches: Transient vs. steady state measurements. • Application of Wilson plot technique for detail investigation and scale-up considerations. Abstract Stirred single-use bioreactors are present throughout the biopharmaceutical manufacturing of high-value cell products. Lots of work has been done determining relevant process engineering parameters to enable reliable scale-up on a physical foundation. However, heat transfer capabilities of such systems are not well described, yet. As processes are further intensified and microbial hosts offer interesting opportunities, heat transfer becomes increasingly relevant. The present work gives a comprehensive overview on the specific implications of a stirred, 50 L, single-use bioreactor by means of heat transfer. This includes the dependency of overall heat transfer coefficients on process parameters, i.e. stirring frequency and jacket flow, with a corresponding statistical analysis. Experimental results are compared against a theoretical approach, based on Nu numbers. Although a systematic discrepancy on the description of the jacket-side heat transfer is present, there already is a good agreement between experimental and literature derived values. The jacket-side impact on the overall performance is then analyzed in detail by the Wilson plot method, underlining the major influence of this aspect. Since the polymer-film layer represents a significant feature of single-use bioreactors, this effect has been investigated for two film types as well. Finally, a first comparison of the 50 L against a 200 L bioreactor was carried out to identify scale-dependencies. [ABSTRACT FROM AUTHOR]

In recent years, single-use bioreactors have been increasingly used in the pharmaceutical industry. However, no direct comparison between single-use and resuable bioreactors have been done at industrial scale. So, the aim of this work is to critically compare hydrodynamics in a Cultibag STR (SUB) and a reusable bioreactor (SSB). The operating conditions and comparison criteria have been chosen in relation to adherent cell cultures. The flow structure and the spatial distribution of mechanical stress were characterized using CFD. A CFD-based compartment model was used to compare mixing and, finally, the trajectories of several particles was simulated with a stochastic model to obtain circulation and residence time distributions in zones of high mechanical stress. Significant differences in terms of hydrodynamics are observed between the two bioreactors. Because of the absence of baffles, the flow in the single-use bioreactor is highly tangential compared to the SSB. As a consequence, the mixing time in the SUB is 2.5 times longer. In the case of adherent animal cell cultures, mechanical stress is usually considered as the main limiting factor related to hydrodynamics. So, the spatial and temporal distribution of mechanical stress have been compared. The results show that the cells grown in the SUB bioreactor will be not only more frequently exposed to high mechanical stress, but also over a longer period. [ABSTRACT FROM AUTHOR]

The knowledge of a volumetric liquid-side mass transfer coefficient (kLa) characterizing the oxygen transfer in bioreactor working at defined operating parameters, is a fundamental principle for establishing the aeration strategy for aerobic bioprocesses. The design of experiments (DoE) methodology has been applied for distinguishing relevant from irrelevant operating parameters, and for prediction of characteristics of oxygen mass transfer effects, in the whole range of values of operating parameters accessible in setup of ReadyToProcess WAVE™ 25 (WAVE 25; GE Healthcare) bioreactor equipped with 2 dm³ disposable culture bag. Due to DoE-aided analysis, rocking speed (ω), rocking angle (α) and volumetric flow of gas phase through the culture bag (QG) have been indicated as the operating parameters robustly impacting on the value of the k[sub L]a coefficient. All relevant operational parameters, i.e. ω, α a and QG, exerted monotonically increasing influence on kLa . Otherwise, the influence of volume of liquid poured into culture bag (VL) and oxygen partial pressure in applied gas phase (p1) on kLa proved to be negligible. Two original correlations have been proposed to generalize the experimental results and to estimate the kLa coefficient values possible to be reached in the WAVE 25: the dimensional correlation defining the kLa coefficient, as well as the dimensionless correlation that defines Sherwood number and integrating the originally-defined Reynolds number for the liquid phase that is subjected to wave-induced mixing. The validity of results predicted by both correlations has been verified by acceptable level of the relative errors. [ABSTRACT FROM AUTHOR]

Stirred tank single-use bioreactors have proven their capability to successfully replace their stainless-steel counterparts in the biopharmaceutical industry. To date, however, only a five-fold volume expansion is achievable in a single-use stirred tank bioreactor, which in turn, necessitates intermediate equipment to scale up the culture to production volume. This study introduces a two-chamber single-use bioreactor that is capable of cell culture expansion from 1 to 50 L. The prototype is a proof of concept that can help users save costs of purchase and qualification of equipment, reduce factory footprint, and reduce the risk of contamination during culture transfer from one intermediate bag to another. The prototype is made of two chambers of different volumes, interconnected as a single, closed system. The design and construction of the prototype is described in detail and results from the engineering characterization (e.g., mixing time, power input per unit volume, and oxygen mass transfer coefficient) are reported for both chambers. The results are in good agreement with general criteria proposed in literature for bioreactor design and with published data for commercially available bioreactors. Further, the concept is not limited to the two-chamber design presented in this study; additional chambers can be integrated along with different volumes, geometries, mixing, sparging, and heating approaches. [ABSTRACT FROM AUTHOR]

Clostridium tetani, a spore-forming anaerobic bacterium, and a casein-based semisynthetic medium were used to produce tetanus toxin in this study. N-Z-Case TT (casein hydrolysate) solution and glucose stock media were mixed and autoclaved, which resulted in tetanus toxin expression. The toxin was expressed when the N-Z-Case TT solution reacted with the glucose stock at a high temperature, creating an adequate amount of Maillard reaction products (MRPs). After accumulating in C. tetani cells, tetanus toxin was secreted into the medium when cell lysis was induced by surface aeration. C. tetani was cultivated and tetanus toxin was expressed in a single-use bioreactor, which produced 80 Lf/mL of tetanus toxin in a medium with MRPs. While using the correct medium to induce tetanus toxin was important, other factors played a part in achieving the desired concentration of the toxin, including the medium processing and culture methods inside the bioreactor. Tetanus toxoid with a purity level greater than 2,500 Lf/mgPN was obtained by detoxifying and purifying the toxin recovered from the fermenter or single-use bioreactor. A single-use bioreactor could be used in a limited space without the need for constructing a large scale production facility, to produce the tetanus toxoid antigen for clinical trials. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-WheelTM technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals. [ABSTRACT FROM AUTHOR]

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System™ (PBS) employs the Air-wheel™, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The ka value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a ka value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10 cells/mL) andwas similar to the STR (9.7 × 10 cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system. [ABSTRACT FROM AUTHOR]

A multitude of heterologous proteins is needed in pharmaceutical research. These have to be produced in robust and flexible cultivation systems to allow a fast and cost-effective handling. Moreover, these systems should be scalable and meet quality and regulatory requirements. The baculovirus expression vector system propagated on High Five insect cells is a popular choice for this purpose. For this expression system an efficient, robust and scalable process was developed based on a single-use technology and a medium replacement strategy with cell retention. It is shown that a medium replacement in the Wave bioreactor is feasible and can be scaled up to a 10-L scale without much effort. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Contamination-free process measurement is essential for efficient monitoring of fermenters. This contribution introduces the use of high-frequency sensors for the contactless measurement of the permittivity and conductivity of fermentation media. As microwaves can penetrate through plastic materials without much attenuation, it is possible to achieve measurement setups that can measure the media under test without any direct contact and with long-term stability. The sensor can be installed directly to the outer surface of the fermenter or can be clamped onto tubing. [ABSTRACT FROM AUTHOR]

This study presents the concept of the travelling wave single-use bioreactor, based on an orbitally shaken, annular-shaped vessel. A numerical model was developed for early design studies in order to reduce the number of prototypes. The flow characteristics in two different vessel shapes were investigated. It was shown that the orbital motion combined with the toroidal shape of the bioreactor create the desired wave characteristics in the contents of the vessel. [ABSTRACT FROM AUTHOR]

Although vertical column, flat-plate, tubular, bubble column and airlift photobioreactors are widely used for the cultivation of diatoms, it has been shown that bubble-induced hydrodynamic stress created in these types of bioreactor can damage cells. Therefore, three single-use surface-aerated bag bioreactors, known for their outstanding results in cultivating shear-sensitive mammalian cells, were for the first time investigated for their suitability in growing the model microalgae Phaeodactylum tricornutum. All of the systems, which were additionally aerated with CO2 and equipped with illumination systems providing different light qualities, guaranteed a 22- to 43-fold increase in cell density within seven days, without any addition of cell protection agents or changes in cell morphology. Maximum cell density and dry biomass were achieved in the orbitally shaken 2D-bag by using cool-white fluorescent tubes. [ABSTRACT FROM AUTHOR]

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10-15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na, osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors. [ABSTRACT FROM AUTHOR]

Understanding the hydrodynamic conditions in bioreactors is of utmost importance for the selection of operating conditions during cell culture process development. In the present study, the two-phase flow in the lab-scale single-use bioreactor Xcellerex TM XDR-10 is characterized for working volumes from 4.5 L to 10 L, impeller speeds from 40 rpm to 360 rpm, and sparging with two different microporous spargers at rates from 0.02 L min − 1 to 0.5 L min − 1 . The numerical simulations are performed with the one-way coupled Euler–Lagrange and the Euler–Euler models. The results of the agitated liquid height, the mixing time, and the volumetric oxygen mass transfer coefficient are compared to experiments. For the unbaffled XDR-10, strong surface vortex formation is found for the maximum impeller speed. To support the selection of suitable impeller speeds for cell cultivation, the surface vortex formation, the average turbulence energy dissipation rate, the hydrodynamic stress, and the mixing time are analyzed and discussed. Surface vortex formation is observed for the maximum impeller speed. Mixing times are below 30 s across all conditions, and volumetric oxygen mass transfer coefficients of up to 22.1 h − 1 are found. The XDR-10 provides hydrodynamic conditions which are well suited for the cultivation of animal cells, despite the unusual design of a single bottom-mounted impeller and an unbaffled cultivation bioreactor. [ABSTRACT FROM AUTHOR]

• Development of a bioreactor with low-cost materials. • Roots distribute to more than 70 % of the liquid culture volume without using any support matrix. • It is possible to use a set of bioreactors in a modular design for mass production of adventitious and hairy roots. Bioreactors are widely used for industrial production of therapeutic products. In this study, a disposable bubble column bioreactor made from flexible plastic films and cheap accessories was developed and its applicability for plant root culture was tested. Hypericum perforatum adventitious roots were cultivated in a set of 15, 30, and 60-L bioreactors for a four weeks period and 14.4, 11, and 10 g dry roots per liter were harvested respectively. Appropriate air flow pattern caused the roots to distribute to more than 70 % of the liquid culture volume without using any support matrix. The packing densities showed 30 % variation at different location of root biomass. Determination of hypericins, total phenols, and total flavonoids indicated some variation throughout the bioreactor, however, the average of them resembled the roots cultivated in flasks. Appropriate bioreactors remain an obstacle in commercialization of plant root cultures and the developed bioreactor seems appropriate for mass production of some adventitious and hairy roots in a modular design. [ABSTRACT FROM AUTHOR]

The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector-manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. Packed-bed or fixed-bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid-1900s. However, the world's first single-use, fully integrated, high cell density, fixed-bed bioreactor was launched only approximately a decade ago. By now, several single-use, fixed-bed technology solutions have been developed in a small scale. Scaling-up the manufacturing can be challenging and for commercial-scale manufacturing, there is practically only one single-use, good manufacturing practice-compliant option available. This study reviews the latest, fully disposable, fixed-bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost-related topics. It is predicted that single-use, fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields., (© 2020 Wiley-VCH GmbH.)

Human mesenchymal stem cells (hMSCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction or inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hMSC-based therapies, in vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible, and economic in vitro expansion of hMSCs for autologous and allogeneic therapies can be problematic because the cell material is restricted and the cells are sensitive to environmental changes. It is beneficial to collect detailed information on the hydrodynamic conditions and cell growth behavior in a bioreactor system, in order to develop a so called "Digital Twin" of the cultivation system and expansion process. Numerical methods, such as Computational Fluid Dynamics (CFD) which has become widely used in the biotech industry for studying local characteristics within bioreactors or kinetic growth modelling, provide possible solutions for such tasks.In this review, we will present the current state-of-the-art for the in vitro expansion of hMSCs. Different numerical tools, including numerical fluid flow simulations and cell growth modelling approaches for hMSCs, will be presented. In addition, a case study demonstrating the applicability of CFD and kinetic growth modelling for the development of an microcarrier-based hMSC process will be shown.

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO 2 ) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO 2 was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k L a values of O 2 (k L a[O 2 ]) and CO 2 [k L a(CO 2 )] in the SS bioreactors as a scale-up factor for dCO 2 reduction. The k L a ratios [k L a(CO 2 )/k L a(O 2 )] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k L a ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO 2 trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k L a ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k L a ratios for the SUBs were evaluated and conditions that provided k L a ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k L a ratio is a powerful scale-up factor useful to control dCO 2 during fed-batch cultures., (© 2020 American Institute of Chemical Engineers.)