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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 7.2·10^6 cells/mL and maximum virus titers of 1.7·10^11vg/mL 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 4.8·10^15 vg that, depending on the therapy, can be sufficient for the treatment of a single patient.

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.

In response to the FDA's call for applying Quality by Design (QbD) to the manufacturing process, the biopharmaceutical industry has invested extensively into the monitoring and controlling of product quality attributes for bioprocesses. To assure the safety and efficacy of the drug product, defining critical quality attributes (CQA) and understanding their correlation with critical process parameters (CPP) becomes vitally important. In this work, a liquid chromatography-mass spectrometry based multi-attribute method (MAM) has been applied to the monitoring and trending of multiple CQAs of a monoclonal antibody product. To the best of our knowledge, this is the first demonstration of applying MAM to both a 3-liter development mini-bioreactor (3 L bioreactor) and a 2000-liter GMP single use bioreactor (2000L SUB). MAM was proven not only to be a great analytical tool for monitoring product quality attributes throughout the time course of the cell culture process, it could also provide critical product quality information in order to understand any potential process performance differences during scale-up and/or technology transfer. The successful monitoring and trending of the multiple CQAs throughout the 17-day cell culture process lays a solid foundation for possible real time in-process control and release of biotherapeutics using MAM in the future.

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.

A group of the DECHEMA working group “Single-Use Technology in Biopharmaceutical Manufacture” is investigating the potential use of single-use systems for cellular agricultural applications. The preliminary results are summarized in this article.

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.

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.

Aim of this work was to evaluate stirred, single-use bioreactors by means of their heat transfer capabilities. Although this bioreactor concept is increasingly adapted by the biopharmaceutical industry, this technical aspect is currently underrepresented in the literature. In contrast to conventional, stainless-steel bioreactors, single-use systems provide some inherent properties that possibly affect heat transfer, such as the polymer film layer of the cultivation chamber and a limited heat transfer area. Therefore, commercially available reaction systems should be investigated. To assess the relevant systems, an adequate experimental procedure was developed. Generally, two types of methods are available: transient experiments, i.e., heating and cooling curves, can directly be applied to jacketed, stirred bioreactors with minimal modification. However, results derived from this approach are more difficult to evaluate, because of the dynamic system behavior. Secondly, steady state methods can be applied, which require an additional heat source. Often electrical heaters are used. In this work, a chemical alternative was established and applied up to a scale of 200 L: the exothermic decay of hydrogen peroxide. Another published work focused on a comprehensive study of thermal properties of stirred single-use bioreactors, where especially transient and steady state experiments were carried out in parallel and set into relation. Finally, a full-system heat transfer model of a stirred 500 L bioreactor was set up, considering all relevant peripheral components required for heat transfer. Single-use bioreactors are mainly used for mammalian cell cultures. The challenging requirements towards the reaction equipment imposed by microbial cultures can hardly be fulfilled by current systems. Nevertheless, these applications are industrially relevant and could generally benefit from the technology. One of the main challenges lies in the metabolic heat produced by the microbial cells. To investigate the biological side, a similar experimental approach was applied to E. coli fed-batch processes, but in a 5 L glass bioreactor. The present work, therefore, represents a contribution to both, the engineering characterization of single-use bioreactors and the related application of calorimetric methods to microbial processes., Ziel dieser Arbeit war es, ger��hrte Einweg-Bioreaktoren hinsichtlich ihrer W��rme��bertragungsf��higkeit zu bewerten. Obwohl dieses Bioreaktorkonzept zunehmend von der biopharmazeutischen Industrie adaptiert wird, ist dieser technische Aspekt derzeit in der Literatur unterrepr��sentiert. Im Gegensatz zu herk��mmlichen Bioreaktoren aus Edelstahl bieten Single-Use-Systeme einige inh��rente Eigenschaften, die m��glicherweise die W��rme��bertragung beeinflussen, wie die Polymerfolienschicht der Kultivierungskammer und eine begrenzte W��rme��bertragungsfl��che. Daher sollten kommerziell erh��ltliche Reaktionssysteme untersucht werden. Zur Bewertung der relevanten Systeme wurde ein ad��quates experimentelles Vorgehen entwickelt. Grunds��tzlich stehen zwei Arten von Methoden zur Verf��gung: Transiente Experimente, d. h. Aufheiz- und Abk��hlkurven, k��nnen mit minimalen Modifikationen direkt auf ummantelte, ger��hrte Bioreaktoren angewendet werden. Ergebnisse aus diesem Ansatz sind jedoch aufgrund des dynamischen Systemverhaltens schwieriger zu bewerten. Zweitens k��nnen station��re Verfahren angewendet werden, die eine zus��tzliche W��rmequelle ben��tigen. H��ufig werden elektrische Heizungen verwendet. In dieser Arbeit wurde eine chemische Alternative etabliert und bis zu einem Ma��stab von 200 L angewendet: der exotherme Zerfall von Wasserstoffperoxid. Eine weitere ver��ffentlichte Arbeit konzentrierte sich auf eine umfassende Untersuchung der thermischen Eigenschaften von ger��hrten Einweg-Bioreaktoren, wobei insbesondere transiente und station��re Experimente parallel durchgef��hrt und in Beziehung gesetzt wurden. Schlie��lich wurde ein Gesamtsystem-W��rme��bertragungsmodell eines ger��hrten 500 L Bioreaktors unter Ber��cksichtigung aller relevanten peripheren Komponenten, die f��r die W��rme��bertragung erforderlich sind, erstellt. Einweg-Bioreaktoren werden haupts��chlich f��r S��ugerzellkulturen verwendet. Die hohen Anforderungen an die Reaktionsapparatur, die mikrobielle Kulturen stellen, k��nnen mit aktuellen Systemen kaum erf��llt werden. Dennoch sind diese Anwendungen industriell relevant und k��nnten generell von der Technologie profitieren. Eine der Hauptherausforderungen liegt in der von den mikrobiellen Zellen produzierten Stoffwechselw��rme. Um die biologische Seite zu untersuchen, wurde ein ��hnlicher experimenteller Ansatz auf E. coli Fed-Batch-Prozesse angewendet, jedoch in einem 5 L Glasbioreaktor. Die vorliegende Arbeit leistet somit einen Beitrag sowohl zur ingenieurtechnischen Charakterisierung von Einweg-Bioreaktoren als auch zur damit verbundenen Anwendung kalorimetrischer Methoden auf mikrobielle Prozesse.

In recent years, the use of hMSCs, which may be isolated from adipose tissue among others, for the treatment of diseases has increased significantly. The cell quantities required for such therapeutic approaches, between 1012 and 1013, have thus far been predominantly produced using commercially available multi-tray systems, such as the Cell Factory (Thermo Fisher Scientific) or HYPERStack (Corning), which can be purchased with up to 40 layers. However, the handling of these planar multilayer systems is difficult, and process monitoring opportunities remain limited. Here, automated stirred single-use bioreactors provide a viable alternative to the time-consuming multiplication of cells using such planar systems, while still managing to achieve the desired clinically relevant quantities. In these stirred single-use systems, adherent cells are predominantly cultivated in suspension up to pilot scale using carrier materials, also referred to as microcarriers (MCs).This chapter describes the steps which need to be realized to guarantee successful hMSC expansion within a stirred single-use bioreactor (Eppendorf's BioBLU® 0.3c) operated using MCs under serum- and xeno-free conditions at benchtop scale. The cultivations were performed using an immortalized human adipose-derived mesenchymal stem cell (hASC) line, hence referred to as hASC52telo, and a new chemically defined, xeno-free medium, hence referred to as the UrSuppe formulation. Spinner flask cultivations were performed under comparable process conditions.

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 kL a values of O2 (kL a[O2 ]) and CO2 [kL a(CO2 )] in the SS bioreactors as a scale-up factor for dCO2 reduction. The kL a ratios [kL a(CO2 )/kL a(O2 )] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the kL 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 dCO2 trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This kL a ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The kL a ratios for the SUBs were evaluated and conditions that provided kL 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 kL a ratio is a powerful scale-up factor useful to control dCO2 during fed-batch cultures.

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.

Clavulanic acid (CA) is a β-lactam antibiotic with potent inhibitory activity against β-lactamase enzymes, which are responsible for the antibiotic resistance phenomenon in several pathogenic bacteria. CA is a secondary metabolite of pharmaceutical and industrial interest naturally produced by the filamentous Gram-positive bacterium Streptomyces clavuligerus (S. clavuligerus) under limited nutritional conditions. CA has limited availability in the market, and its high cost is a consequence of the complexity of the production process. It is determined mainly by the low titers of CA obtained in submerged cultivations of S. clavuligerus and the difficulties associated with the down-stream process. Several authors have applied experimental approaches to study the influence of some variables, especially those of nutritional nature, on CA accumulation. Nevertheless, the effect of variables relevant to the bioprocess operation, as the reactor hydrodynamics and shear stress conditions, has not been well explored. Fluxomic approaches have been recently applied to S. clavuligerus aimed to improve the understanding of its metabolism. Such approaches were mostly developed under steady-state assumptions, resulting in a limited comprehension of the metabolism under the dynamic conditions of batch and fed-batch processes. In this thesis, the reconstruction of a new and enhanced genome-scale model of S. clavuligerus, the successful combination of experimental studies in the shake flask and bioreactor scales coupled with constraint-based modeling in pseudo-steady (flux balance analysis) and dynamic (dynamic flux balance analysis) conditions were used as strategies for studying the metabolic response of S. clavuligerus to environmental and nutritional perturbations in connection with CA biosynthesis. Experimental studies in stirred tank and 2-D rocking-motion bioreactors provided valuable information on the strain's metabolic response to environmental conditions, especially regarding the effect of shear forces. Moreover, the experimental data obtained allowed to test different in silico scenarios by using constraint-based modeling with a new and enhanced reconstruction of a genome-scale metabolic network of S. clavuligerus, aimed to understand the carbon fluxes distribution during the different environmental conditions attained during the cultivations. The use of constrained-based modeling under pseudo-steady state conditions (Flux Balance Analysis, FBA) and dynamic conditions (Dynamic Flux Balance Analysis, DFBA) allowed to explain the role of primary metabolism and revealed the dynamics of intracellular carbon fluxes distribution during CA biosynthesis. Furthermore, the in silico simulation of metabolic scenarios and experimental testing showed that fed-batch operation with glutamate supplementation is a favorable condition for increasing the CA production. Potential genetic engineering targets were identified and evaluated in silico, aiming to improve the CA titers in S. clavuligerus cultures. This is the first work considering the cultivation dynamics on experimental and in silico studies of S. clavuligerus metabolism.

The optimization of operating conditions is vital for cell culture process development. In the present study, the pilot-scale single-use bioreactor XcellerexTM XDR-200, which is typically used in cell culture scale-up, is investigated. This unbaffled reactor is equipped with an off-centered, bottom mounted impeller, and has a maximum working volume of 200 L. The flow field for this sparsely investigated bioreactor configuration is studied with computational fluid dynamics. Moreover, numerical simulations of the mixing time and the volumetric oxygen mass transfer coefficient for seven different test conditions are validated against experimental data. Mixing times across all tested conditions are found to remain below 30 s, and the volumetric oxygen mass transfer coefficient is between 2.0 to 20.0 h−1, which is a typical range for animal cell culture processes. These values of the oxygen transfer coefficient are found for as low as 0.004 to 0.125 L L−1 min−1 sparging rates with the microporous sparger. The hydrodynamic stress remains below critical values for cell damage for all conditions investigated. Vortex formation is suppressed in the bioreactor even though it is unbaffled due to off-centered impeller. Across the test conditions, the highest investigated impeller speed of 200 rpm is the most favorable due to fast mixing and high oxygen transfer.

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.

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 kLa coefficient. All relevant operational parameters, i.e. ω, α 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.

The flow characteristics e.g. velocity and velocity gradients in a conventional stirred sMBR with in-line tubular membrane module for integrated production and recovery of value-added material, are studied. Considering a hybrid vessel, the flow characteristics are conflicting parameters, viz. high shear is required for membrane walls and low shear required for microbial cells. Attempt is made to find a range of parameters that are balanced against known critical values. The task is resolved by numerical solution of a theoretical model of dual flat-blade impeller Biostat® 5 L (T = 0.16 m, D = 6.6 cm) equipped with tubular membrane module (L = 23 cm, d = 12 mm) operating in non-Newtonian biofluid (flow index range, 0.34 γ ˙ versus input power [ γ ˙ = C ( P / V R ) 1 / 3 ] is confirmed. A range of balanced bulk and wall shear relevant to hybrid operation is determined. In view of the reported scalability of the conventional MBR design and reusable bioreactors, the data could be used for extrapolation.

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.

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.

To ensure high quality output of biotechnological processes, relevant process parameters need to be monitored. As bioprocesses are increasingly executed in single use bioreactors, there is an increasing demand for new sensors applicable to these processes. In this work, we investigate different approaches for continuous non-invasive cell growth monitoring, especially for single use bioreactor applications. Therefore, the permittivity of the cell culture is used as a measure for the biomass. In a first step, a measuring procedure based on the transmission measurement of an electromagnetic wave is investigated. It appears that the penetration depth of this sensor is not sufficient for a noninvasive measurement through the polymer wall of a single use bioreactor. Therefore, alternative setups based on magnetic induction are investigated. The initial setup is very simple. It consists of a planar coil connected to an impedance analyzer. The coil is attached to the outside of the polymer foil of the single use bioreactor and an impedance spectrum is measured. To evaluate the sensor, E. coli cultivations are performed in a modified cultivation setup, which enables measurements through the polymer foil of a Sartorius BIOSTAT® CultiBag RM, and additionally allows sampling of culture medium for optical density reference measurements. The resonance peak of the coil in the impedance spectrum, is observed as measure for the optical density. Regardless of the simple sensor construction, we found a good correlation between optical density and the damping ratio of the resonance peak. However, the sensor signal shows saturation towards high optical densities. Therefore, an LTCC coil producing a higher magnetic flux density in the culture medium is investigated subsequently. This sensor shows a linear response up to high optical densities, but the sensitivity is reduced compared to the former used coil and therefore scattering of the data is increased. However, to increase the sensitivity, a linear variable differential transformer is realized. Using this setup, the influence of the primary magnetic flux is eliminated from the measuring voltage. This approach delivers the most promising results, as the sensor response is linear up to high optical densities and data scattering is low.

One of the actual challenges in tissue engineering applications is to efficiently produce as high of number of cells as it is only possible, in the shortest time. In static cultures, the production of animal cell biomass in integrated forms (i.e. aggregates, inoculated scaffolds) is limited due to inefficient diffusion of culture medium components observed in such non-mixed culture systems, especially in the case of cell-inoculated fiber-based dense 3D scaffolds, inside which the intensification of mass transfer is particularly important. The applicability of a prototyped, small-scale, continuously wave-induced agitated system for intensification of anchorage-dependent CP5 chondrocytes proliferation outside and inside three-dimensional poly(lactic acid) (PLA) scaffolds has been discussed. Fibrous PLA-based constructs have been inoculated with CP5 cells and then maintained in two independent incubation systems: (i) non-agitated conditions and (ii) culture with wave-induced agitation. Significantly higher values of the volumetric glucose consumption rate have been noted for the system with the wave-induced agitation. The advantage of the presented wave-induced agitation culture system has been confirmed by lower activity of lactate dehydrogenase (LDH) released from the cells in the samples of culture medium harvested from the agitated cultures, in contrast to rather high values of LDH activity measured for static conditions. Results of the proceeded experiments and their analysis clearly exhibited the feasibility of the culture system supported with continuously wave-induced agitation for robust proliferation of the CP5 chondrocytes on PLA-based structures. Aside from the practicability of the prototyped system, we believe that it could also be applied as a standard method offering advantages for all types of the daily routine laboratory-scale animal cell cultures utilizing various fiber-based biomaterials, with the use of only regular laboratory devices.

Single-use bioreactors have increasingly been used in recent years, for both research and development as well as industrial production, especially in mammalian cell-based processes. Among the numerous single-use bioreactors available today, wave-mixed bags and stirred systems dominate. Wave-mixed single-use bioreactors are the system of choice for inoculum production, while stirred single-use bioreactors are most often preferred for antibody expression. For this reason, the present chapter describes protocols instructing the reader to use the wave-mixed BIOSTAT® RM 50 for cell expansion and to produce a monoclonal antibody (mAb) in Pall's Allegro™ STR 200 at pilot scale for the first time. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing a recombinant immunoglobulin G (IgG).

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (kLa) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. The fed-batch process utilizes a recombinant variant of the mammalian cell line, Chinese hamster ovary (CHO), to express a biosimilar of a therapeutic monoclonal antibody. The perfusion-based process utilizes either an alternating tangential flow filtration (ATF) or a tangential flow filtration (TFF) system for cell retention to cultivate an avian cell line, AGE1.CR.pIX, for the propagation of influenza A virus, H1N1, in high cell density. Based on two example cell cultivations, processes outline the advantages that come with using an orbitally shaken bioreactor for scaling-up a process. The described methods are also applicable to other suspension cell lines.

The increasing importance of viral vaccine manufacturing has driven the need for high cell density process optimization that allows for higher production levels. Vero cells are one of the more popular adherent cell lines used for viral vaccine production. However, production is limited due to the logistical limitations surrounding adherent cell line processes, such as large equipment footprints, time and labor-intensive processes, and larger costs per dose. We have addressed this limitation with the establishment of a viral vaccine production system utilizing the novel single use scale-X™ carbo bioreactor. The unit is compact and is scalable and one of the novel features of the system is the continuous in-line downstream purification and concentration processes associated with the bioreactor vessel. We present the results from a campaign featuring a proprietary Vero cell line for production of a live recombinant Vesicular stomatitis virus vaccine that features the Lassa Fever virus glycoproteins. Metabolite analyses and viral yield comparison between traditional flasks, cell factories, and the scale-X carbo bioreactor were performed, and on average, the single use bioreactor produced 2–4 logs higher titers per surface area, approximately 5 × 1010 pfu/cm2, compared to classical flatstock, 2.67 × 106 pfu/cm2, and cell factories production, 5.77 × 108 pfu/cm2. Overall, we describe a novel bioreactor platform that allows for a cost-efficient and scalable process for viral vaccine production.

Магістерська робота на здобуття освітньо-кваліфікаційного рівня «магістр» на тему «Дослідження удосконаленої конструкції перемішуючого пристрою для single­use біореактора» /НТУУ «КПІ імені Ігоря Сікорського». Керівник к.т.н,, Костик С.І. Виконавець Яремчук М.М., студент групи БІ-81мп, Текстова частина даного магістерського проекту складається з 83 сторінок. З яких 26 сторінок складають розрахунки що підтверджують надійність та працездатність апарату, 11 сторінок економічних розрахунків та 49 сторінки теоретичної частини. Пояснювальна записка також містить 29 зображень 13 таблиць та 3 додатки. Метою даного проекту є розробка вдосконаленого перемішуючого пристрою для Single-use біореактора, який би мав вищу ефективність меншу ціну та носив покращенні техніко-економічні властивості порівняно з існуючими перемішуючими пристроями для біореакторів. Запропонована конструкція є монолітною, забезпечує високу якість Культивування. Даний пристрій може бути встановлений практично у кожному |біореакторі й не вимагає особливого до себе відношення. Використана методика розробки та доведення ефективності перемішуючого шжстрою може бути використана при проектуванні нових конструкцій механічних перемішуючих пристроїв. Master's work on obtaining the educational-qualification level "Master's degree" on "Research of advanced design of mixing device for single-use bioreactor" / NTUU "KPI named by Igor Sikorsky". Scientific director PhD, Associate Professor of Engineering, Kostyk S.I. Performer Yaremchuk M.M., student of the group BI-81mp, The textual part of this master's project consists of 83 pages. Of which 26 pages are calculations confirming the reliability and efficiency of the machine, 11 pages of economic calculations and 49 pages of theoretical part. The explanatory note also contains 29 images of 13 tables and 3 appendices. The purpose of this project is to develop an improved mixer for Single-use bioreactor, which would be less cost effective and have improved technical and economic properties compared to existing bioreactor mixers. The proposed construction is monolithic, provides high quality cultivation. This tdevice can be installed in virtually every bioreactor and does not require special treatment. The method used to design and prove the effectiveness of the mixing device can be (used in the design of new designs of mechanical mixing devices.

In this study, Euler-Euler coupled computational fluid dynamics (CFD), species transport and population balance model (PBM) simulations have been used to model multiphase hydrodynamics and mass (O2) transfer in a single use bioreactor (SUB) for mAb production. In simulations, drag was estimated by Schiller-Neuman equations. O2(g) local volume fraction was estimated via species transport model (STM). The PBM equations were solved using quadrature method of moments (QMOM) to get bubble size distribution (BSD) and thereby the Sauter mean diameter,d32. Also, the mean diameter over surface (d21) and De Brouckere mean (d43) were estimated for calculation of the BSD. The mass transfer coefficient (kLa) was predicted using species transport model (STM) and Higbie’s penetration theory. The experimental estimation of kLa was performed via simplified dynamic pressure method. Total stress (τ) and energy dissipation rate (e) were estimated as 34.17 Pa and 1.352 m2.s−3, respectively. Calculations of the d32, d21 and d43 indicate that the BSD becomes more dispersed with increase in agitation speed at all aeration rates. Further, the kLa values of STM were within 0.47% to 4.21% of experimental data. In summary, with error between 4 and 10%, the proposed approach offers better visualization of O2 transport mechanism across gas–liquid interface than conventional approaches.

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.

Bioreactor, the womb for human stem cells, plays a vital role in enhancing its proliferation under controlled environment. Stem cells have the potential to differentiate and replicate it-self to further grow human tissues and organs. Bioreactor nurtures stem cells by providing nutritious environment and peerless oxygen flow inside the cell bag without obstructing the sheer force of the fluid or gas bubbles. The present paper unveils the design and development of bioreactor using the gyrator motion mechanism. This high functional bioreactor provides automation system that enhances the environment and serves excellent nutrient distribution to exceed cell growth through oxygen transfer and better mixing. One of the best characteristic of this bioreactor is that it requires no cleaning or sterilization as it is disposable. Compared to other bioreactors such as spinner flask type, rotating-wall type and the hollow fibre type, the proposed bioreactor is more convenient, easy to operate and maintain with more flexibility. Proposed bioreactor system operates in conjunction with mechanism of external gyrating motion that uses gravity in a natural way to make the flow circular inside it. The simultaneous mixing motion base is extremely suitable to grow stem cells. This bioreactor can be easily placed in an incubator and further addition or sampling is viable without the use of laminar flow cabinet.

To ensure high quality output of biotechnological processes, relevant process parameters need to be monitored. As bioprocesses are increasingly executed in single use bioreactors, there is an increasing demand for new sensors applicable to these processes. In this work, we present a low-cost sensor system for continuous non-invasive cell growth monitoring, especially for single use bioreactor applications. The system consists of a planar coil connected to a low cost network analyzer. The coil is attached to the outside of the polymer foil of the single use bioreactor and an impedance spectrum is measured. To evaluate the sensor, E. coli cultivations are performed in a modified cultivation setup, which enables measurements through the polymer foil of a Sartorius BIOSTAT ® CultiBag RM, and additionally allows sampling of culture medium for optical density reference measurements. The resonance peak of the coil in the impedance spectrum, is observed as measure for the optical density. Regardless of the simple sensor construction, we found a good correlation between optical density and the damping ratio of the resonance peak.

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.

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.

Single‐use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO‐K1, and CHO‐DP12, cultured in media conditioned in an older single‐use bioreactor (SUB) film (F‐1) and a newer generation film (F‐2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F‐1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F‐2 film. Proteomic profiling of CHO cells cultured in the F‐1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F‐2 films when compared to F‐1 films including bDtBPP, potentially explaining improved CHO cell growth in F‐2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess. Science Foundation Ireland Irish Industry Technical Group (Allergan Pharmaceuticals Ireland, BioMarin Manufacturing Ireland Ltd., Eli Lilly and Company, Genzyme Ireland Ltd. A Sanofi Company, Janssen Biologics, MSD and Pfizer Ireland Pharmaceuticals) Dublin City University

Erworben im Rahmen der Schweizer Nationallizenz (http://www.nationallizenzen.ch), Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term “disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.

Heart valve tissue engineering offers the promise of improved treatments for congenital heart disorders; however, widespread clinical availability of a tissue engineered heart valve (TEHV) has been hindered by scientific and regulatory concerns, including the lack of a disposable, bioreactor system for nondestructive valve seeding and mechanical conditioning. Here we report the design for manufacture and the production of full scale, functional prototypes of such a system. To evaluate the efficacy of this bioreactor as a tool for seeding, ovine aortic valves were decellularized and subjected to seeding with human mesenchymal stem cells (hMSC). The effects of pulsatile conditioning using cyclic waveforms tuned to various negative and positive chamber pressures were evaluated, with respect to the seeding of cells on the decellularized leaflet and the infiltration of seeded cells into the interstitium of the leaflet. Infiltration of hMSCs into the aortic valve leaflet was observed following 72 h of conditioning under negative chamber pressure. Additional conditioning under positive pressure improved cellular infiltration, while retaining gene expression within the MSC-valve interstitial cell phenotype lineage. This protocol resulted in a subsurface pilot population of cells, not full tissue recellularization. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 249-259, 2017.

Studies of the extractable profiles of bioprocessing components have become an integral part of drug development efforts to minimize possible compromise in process performance, decrease in drug product quality, and potential safety risk to patients due to the possibility of small molecules leaching out from the components. In this study, an effective extraction solvent system was developed to evaluate the organic extractable profiles of single-use bioprocess equipment, which has been gaining increasing popularity in the biopharmaceutical industry because of the many advantages over the traditional stainless steel-based bioreactors and other fluid mixing and storage vessels. The chosen extraction conditions were intended to represent aggressive conditions relative to the application of single-use bags in biopharmaceutical manufacture, in which aqueous based systems are largely utilized. Those extraction conditions, along with a non-targeted analytical strategy, allowed for the generation and identification of an array of extractable compounds; a total of 53 organic compounds were identified from four types of commercially available single-use bags, the majority of which are degradation products of polymer additives. The success of this overall extractables analysis strategy was reflected partially by the effectiveness in the extraction and identification of a compound that was later found to be highly detrimental to mammalian cell growth. LAY ABSTRACT: The usage of single-use bioreactors has been increasing in biopharmaceutical industry because of the appealing advantages that it promises regarding to the cleaning, sterilization, operational flexibility, and so on, during manufacturing of biologics. However, compared to its conventional counterparts based mainly on stainless steel, single-use bioreactors are more susceptible to potential problems associated with compound leaching into the bioprocessing fluid. As a result, extractable profiling of the single-use system has become essential in the qualification of such systems for its use in drug manufacturing. The aim of this study is to evaluate the effectiveness of an extraction solvent system developed to study the extraction profile of single-use bioreactors in which aqueous-based systems are largely used. The results showed that with a non-targeted analytical approach, the extraction solvent allowed the generation and identification of an array of extractable compounds from four commercially available single-use bioreactors. Most of extractables are degradation products of polymer additives, among which was a compound that was later found to be highly detrimental to mammalian cell growth.

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (kL a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h-1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 109 cells L-1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018.

A method for the identification of leachables in chemically defined media for CHO cell culture using dispersive liquid-liquid microextraction (DLLME) and UHPLC-MS is described. A Box-Behnken design of experiments (DoE) approach was applied to obtain the optimum extraction conditions of the target analytes. Performance of DLLME as extraction technique was studied by comparison of two commercial chemically defined media for CHO cell culture. General extraction conditions for any group of leachables, regardless of their specific chemical functionalities can be applied and similar optimum conditions were obtained with the two media. Extraction efficiency and matrix effects were determined. The method was validated using matrix-matched standard calibration followed by recovery assays with spiked samples. Finally, cell culture media was incubated in 7 single use bioreactors (SUBs) from different vendors and analysed. TBPP was not detected in any of the samples, whereas DtBP and TBPP-ox were found in all samples, with bDtBPP detected in six SUBs. This method can be used for early identification of non-satisfactory SUB films for cultivation of CHO cell lines for biopharmaceutical production.

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 themeasurement range of the torque meter. A comparison of dimensionless power numbers determined for a reference Rushton turbine stirrer (N-P = 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 > 10(4). Power numbers of between 0.3 and 4.5 (for Re = 10(4)) 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.

In this work, we present a low-cost sensor system for continuous non-invasive cell growth monitoring, especially for single use bioreactor (SUB) applications. The sensor system is based on a differential transformer. Using this differential setup, the influence of the primary magnetic flux is eliminated from the measuring signal, enabling highly sensitive non-invasive detection of permittivity changes in the culture medium. To evaluate the sensor, E. coli cultivations are performed and the cell density is measured through the polymer foil of a SUB. We found a linear dependency with low data scattering between measuring signal and cell density.

Single-use bioreactors (SUBs) show clear benefits compared to the traditional fixed glass and steel bioreactor equipment used for bioprocessing. Reduction of validation efforts, sterilization in advance, and ease of use have a positive impact on costs and timelines for the development of biopharmaceuticals. Several types of SUBs have been introduced, where rocking-type systems and tank liners are predominantly used. Rocking-type bioreactors are restricted in their use up to 100 l working volumes, whereas the stirred SUBs can be applied in a range from 50 to 2000 l. The rocking-type bioreactors are restricted in use to mammalian cell cultures. To date, stirred-type SUBs have only been applied for mammalian cell culture as well. In both Chinese hamster ovary (CHO) and PER.C6® cultures, these bioreactors result in process performance comparable to the traditional glass or steel bioreactors, and are applied with high-intensity cell-culture processes (XD® process) as well. A new type of rocking bioreactor, the CELL-tainer®, is capable of generating high mass-transfer coefficients and opens up the potential of application of SUBs in high-intensity cell cultures as well as in microbial cultures.

Applying disposable, single-use bioreactors to bioprocessing has triggered a revolution of change to traditional stainless-steel bioprocess workflow. Single use provides a cheaper, faster, and lower-cost solution than stainless steel. Single-use bioreactors have been successfully applied in discovery and preclinical biopharma applications to speed process development and material generation. The combination of continually rising upstream titers with improved single-use technology robustness is driving the expansion of single-use bioreactors into commercial manufacturing. This enables the strategy for future modular facilities with a scale-out rather than scale-up approach. Despite this success, a range of ongoing challenges is being targeted by the industry single-use community, including lowering the supply chain risk, standardizing testing protocols, expanding plug-and-play capability, improving sensor systems, and lowering bag failure. Improving these issues in coming years will reduce the barriers to single-use bioreactor implementation and continue to broaden expansion into commercial biologics, vaccines, and cell therapy manufacturing.

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.

Background & Aim With the recent successes of autologous immunotherapy, with 2 therapeutic products approved by the Food & Drugs Administration (FDA) since 2017 and more in the clinical pipeline, there is a need for cell and gene therapy companies to transition from lab scale production to mass manufacture. Traditionally, bioreactors have been routinely used in process development and scale up manufacturing of biologics. However, such bioreactor systems are not suitable for immunotherapy production as, beyond standard external factors such as shear stresses, immune cells are also sensitive to factors such as density and require cell-to-cell contact to proliferate well. Methods, Results & Conclusion A novel single use bioreactor for non-adherent cell culture has been developed in SIMTech (Patent Application WO2018/182533 A1) that allows for culturing cells at constant density while increasing volume of culture media to support cell proliferation. The bioreactor features two different chambers, one for media reserves and the other for cells. The two chambers are separated by a membrane that allow for fluidic exchange between these chambers. The cell chamber has a built-in expansion mechanism that can be adjusted as cells proliferate, thus allowing the user to adjust at any time point and maintain consistent culture density. The bioreactor has been validated with Jurkat cell line and T cells derived from commercial PBMCs with promising results; it was observed that the cultured cells exhibited high viability and selectivity. Jurkat cells had a 240-fold increase in cell number after 14 days in culture while T cells (from commercial PBMCs), had a 47-fold increase in cell number after 21 days in culture. A direct comparison study (1 bioreactor vs multiple 24-well plates for the same increase in cell number) also demonstrated a significant reduction in protocol steps and culture handling time. The bioreactor is currently being tested on donor PBMCs and other cell types, in collaborations with clinicians and cell therapy laboratories, in order to develop a GMP workflow for clinical use.

Background & Aim There is a significant need for efficient systems that can be used to generate primary cells and stem cells which can be readily implemented in research labs and expedite process development studies, pre-clinical testing, and large-scale expansion. Although various platforms for expansion of sufficient cell numbers exist, these systems are rarely comprehensive enough to allow for rapid implementation by researchers. These systems are also not easy to use nor are they robust enough to reproducibly supply high quality cells. A complete system for the generation of sufficient cell numbers that utilizes a bioreactor platform which is robust and easy-to-use is therefore highly desirable. Methods, Results & Conclusion We have previously demonstrated efficient expansion of human mesenchymal stem/stromal (hMSC) in the PadReactor® single-use bioreactor system to the 40 L scale. This system utilized commercially-available, well-characterized cells, specially-formulated growth media and supplements, SoloHill microcarriers, and a single-use bioreactor platform that could be used to generate tens of billions of cells within 7 days of culture. Here we extend these finding by employing these components in a novel bioreactor type which contains a bottom-mounted impellor. The Allegro™ STR bioreactor is a new single-use stirred tank bioreactor platform which is scalable, compact, ergonomic, and designed to maximize usability and process assurance. hMSC and medium were cultured at 30 L scale in microcarrier-based, fed-batch cultures in the Allegro STR bioreactor for 5 days. Cells harvested from microcarriers retained critical quality attributes when examined in standard cell characterization assays. The results obtained in this study lay the groundwork for a complete system for efficient generation of high quality cells for process development studies and future large scale cell expansion. Together, the Allegro STR bioreactor and the SoloHill microcarriers provide a practical manufacturing solution for large-scale expansion of adherent cells.

The exploitation of single-use systems (SUS) has become a major subject in the biopharmaceutical industry, covering the entire product life cycle, with applications in discovery, development and through to licensed product. The implementation of SUS is a complex subject and subject to differing interpretations. The experience of SUS implementation in Valneva (Nantes, France) is reported here, reviewed together with other references and our lessons learned to illustrate that implementation must be a structured process with clear aims. Some experience gained is illustrated with a practical example that illustrates the difference in exploitation between a single-use bioreactor and a classical stainless steel bioreactor, showing how using SUS can improve operating efficiency and time.

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 kLa 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 kLa 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 × 106 cells/mL) andwas similar to the STR (9.7 × 106 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.

Graphical abstract, Wave-mixed bioreactors have increasingly replaced stainless steel stirred tank reactors in seed inoculum productions and mammalian cell-based process developments. Pre-sterilized, single-use plastic bags are used for cultivation, eliminating the risk of cross-contamination and cleaning procedures. However, these advantages come with high consumable costs which is the main barrier to more uptakes of the technology by academic institutions. As an academic Core Facility that faces high demand in protein production from insect cells, we have therefore developed a cost-effective alternative to disposable wave bags. In our study we identified: • A re-usable wave shaken polycarbonate bioreactor for protein production in insect cells achieves protein yields comparable to disposable bags. • The advantages of this re-usable bioreactor are low costs, long life cycle, flexible configuration of accessories and convenient handling due to its rigid shape.

The BaySHAKE® single-use bioreactor is based on the motion principle of rotary oscillation of a polyhedral container. This fundamental working principle facilitates mixing and oxygen transfer to animal cell cultures in a simple, robust and cost-efficient single-use system without internal moving parts. In-depth hydrodynamic characterization shows that the bioreactor provides mixing and oxygen transfer in a low-shear environment over a range of scales up to several cubic meters. A biological comparison of novel single-use technology in scales from 50 to 1000 L with the re-usable standard deep-tank technology in scales from 10 to 200 L confirms the technical feasibility and shows good biological equivalence for the production of monoclonal antibodies by CHO cells.