Characterizing the fluid dynamics of the inverted frustoconical shaking bioreactor

The authors conducted a three‐dimensional computational fluid dynamics (CFD) simulation to calculate the flow field in the inverted frustoconical shaking bioreactor with 5 L working volume (IFSB‐5L). The CFD models were established for the IFSB‐5L at different operating conditions (different shaking speeds and filling volumes) and validated by comparison of the liquid height distribution in the agitated IFSB‐5L. The “out of phase” operating conditions were characterized by analyzing the flow field in the IFSB‐5L at different filling volumes and shaking speeds. The values of volumetric power consumption (P/V_L) and volumetric mass transfer coefficient (k_La) were determined from simulated and experimental results, respectively. Finally, the operating condition effect on P/V_L and k_La was investigated. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:478–485, 2018     

Large‐scale culture of a megakaryocytic progenitor cell line with a single‐use bioreactor system

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 (k_La) 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     

Correlation between mass transfer coefficient k<Subscript>L</Subscript>a and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

Background

Among disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen k_La and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal k_La correlation.

Results

A Respiration Activity Monitoring System (RAMOS) was used to measure k_La values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for k_La. In this way, a scale- and volume-independent k_La correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.

Conclusion

The resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/? 30%. To our knowledge, this is the first k_La correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.

     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

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 k_La 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 k_La 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.     

Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor

Optimization of a bioreactor design can be an especially challenging process. For instance, testing different bioreactor vessel geometries and different impeller and sparger types, locations, and dimensions can lead to an exceedingly large number of configurations and necessary experiments. Computational fluid dynamics (CFD), therefore, has been widely used to model multiphase flow in stirred-tank bioreactors to minimize the number of optimization experiments. In this study, a multiphase CFD model with population balance equations are used to model gas–liquid mixing, as well as gas bubble distribution, in a 50 L single-use bioreactor vessel. The vessel is the larger chamber in an early prototype of a multichamber bioreactor for mammalian cell culture. The model results are validated with oxygen mass transfer coefficient (k_La) measurements within the prototype. The validated model is projected to predict the effect of using ring or pipe spargers of different sizes and the effect of varying the impeller diameter on k_La. The simulations show that ring spargers result in a superior k_La compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. In addition, larger impellers are shown to improve k_La. A correlation of k_La is presented as a function of both the reactor geometry (i.e., sparger-to-impeller diameter ratio and impeller-to-vessel diameter ratio) and operating conditions (i.e., Reynolds number and gas flow rate). The resulting correlation can be used to predict k_La in a bioreactor and to optimize its design, geometry, and operating conditions.     

Application of a two-dimensional disposable rocking bioreactor to bacterial cultivation for recombinant protein production

Disposable rocking bioreactors (RBs) are widely employed for cultivation of recombinant mammalian and insect cell lines, although the perception of inadequate mass transfer has prevented their application to bioprocesses based on microbial platforms. In this study, one-dimensional (1D) and two-dimensional (2D) RBs were assessed and compared with the conventional stirred tank reactor (STR) for recombinant therapeutic protein production in Escherichia coli. The comparison involved: (1) physical characterization of oxygen mass transfer efficiency and mixing intensity, (2) growth characteristics in batch cultivation, and (3) culture performance for the production of recombinant protein. Our results show that oxygen mass transfer was comparable between the 1D RB and STR at low working volume (WV), declining linearly with increasing WV, and was highest in the 2D RB for all tested WVs with the maximum mass transfer coefficient (k_La) at 3 L WV. Well mixing behavior was observed in all three systems for water and aqueous carboxymethylcellulose (CMC) solutions. Batch growth characteristics were similar in all bioreactor systems, although metabolite accumulation was significant in the 1D RB. Culture performance for the production of recombinant GST-hCD83ext (glutathione S-transferase-hCD83ext fusion protein) was similar in terms of soluble protein yield and inclusion body formation for all bioreactor systems.     

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool

In this study, we perform mass transfer characterization (k_La) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch‐based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5‐fold resulted in 30% increase in k_La (from 2.3 to 3.0 h^?1) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control. Biotechnol. Bioeng. 2012; 109: 2790–2797. © 2012 Wiley Periodicals, Inc.     

Studies on fluid dynamics of the flow field and gas transfer in orbitally shaken tubes

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50‐mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high‐throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three‐dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (k_La) was determined experimentally and from simulations under various working conditions. We also determined the liquid‐phase mass transfer coefficient (k_L) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of k_L. High oxygen transfer rates, sufficient for supporting the high‐density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192–200, 2017     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping 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 k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La 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 k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La 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_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

Microwell engineering characterization for mammalian cell culture process development

Experimentation in shaken microplate formats offers a potential platform technology for the rapid evaluation and optimization of cell culture conditions. Provided that cell growth and antibody production kinetics are comparable to those found in currently used shake flask systems then the microwell approach offers the possibility to obtain early process design data more cost effectively and with reduced material requirements. This work describes a detailed engineering characterization of liquid mixing and gas–liquid mass transfer in microwell systems and their impact on suspension cell cultures. For growth of murine hybridoma cells producing IgG1, 24‐well plates have been characterized in terms of energy dissipation (P/V) (via Computational Fluid Dynamics, CFD), fluid flow, mixing and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted k_La values varied between 1.3 and 29 h^?1; liquid‐phase mixing time, quantified using iodine decolorization experiments, varied from 1.7 s to 3.5 h; while the predicted P/V ranged from 5 to 35 W m^?3. CFD simulations of the shear rate predicted hydrodynamic forces will not be detrimental to cells. For hybridoma cultures however, high shaking speeds (>250 rpm) were shown to have a negative impact on cell growth, while a combination of low shaking speed and high well fill volume (120 rpm, 2,000 µL) resulted in oxygen limited conditions. Based on these findings a first engineering comparison of cell culture kinetics in microwell and shake flask formats was made at matched average energy dissipation rates. Cell growth kinetics and antibody titer were found to be similar in 24‐well microtiter plates and 250 mL shake flasks. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproducible and comparable to currently used shake flask systems while offering at least a 30‐fold decrease in scale of operation and material requirements. Linked with automation this provides a route towards the high throughput evaluation of robust cell lines under realistic suspension culture conditions. Biotechnol. Bioeng. 2010; 105: 260–275. © 2009 Wiley Periodicals, Inc.     

Monitoring gradient formation in a jet aerated bioreactor

Jet aerated loop reactors (JLRs) provide high mass transfer coefficients (k_La) and can be used for the intensification of mass transfer limited reactions. The jet loop reactor achieves higher k_La values than a stirred tank reactor (STR). The improvement relies on significantly higher local power inputs (～10⁴) than those obtainable with the STR. Operation at high local turnover rates requires efficient macromixing, otherwise reactor inhomogeneities might occur. If sufficient homogenization is not achieved, the selectivity of the reaction and the respective yields are decreased. Therefore, the balance between mixing and mass transfer in jet loop reactors is a critical design aspect. Monitoring the dissolved oxygen levels during the turnover of a steady sodium sulfite feed implied the abundance of gradients in the JLR. Prolonged mixing times at identical power input and aeration rates (～100%) were identified for the JLR in comparison to the STR. The insertion of a draft tube to the JLR led to a more homogenous dissolved oxygen distribution, but unfortunately a reduction of mixing time was not achieved. In case of increased medium viscosities as they may arise in high cell density cultivations, no gradient formation was detected. However, differences in medium viscosity significantly altered the mass transfer and mixing performance of the JLR.     

CFD analysis of the turbulent flow in baffled shake flasks

In this work, computational fluid dynamics (CFD) technique is used to simulate the complicated unsteady-state turbulent flow field formed in baffled flask. The baffled flask shows advantages both in mass transfer capacity and in shear formation in comparison with unbaffled flasks. Detailed investigations of power consumption, mass transfer and shear rate are carried out in baffled flasks under shaking frequencies ranging from 100 rpm to 250 rpm, and filling volumes from 50 mL to 150 mL. The results show that the specific power input and specific interface area are both greatly influenced by shaking frequency and filling volume. For the positive effect of shaking frequency on both mass transfer coefficient (k_L) and specific interface area (a), the volumetric mass transfer coefficient (k_La) increases greatly with shaking frequency. Results also show that filling volume has no significant effect on k_L but negative effect on specific interface area. Shear force formed in baffled flask shows great dependent on shaking frequency, but it is insensitive to the filling volume. Based on these investigations, correlations linking these parameters are proposed. Finally, cultivations of filamentous fungus conducted in unbaffled and baffled flasks validated the simulating results.     

Time efficient way to calculate oxygen transfer areas and power input in cylindrical disposable shaken bioreactors

Disposable orbitally shaken bioreactors are a promising alternative to stirred or wave agitated systems for mammalian and plant cell cultivation, because they provide a homogeneous and well‐defined liquid distribution together with a simple and cost‐efficient design. Cultivation conditions in the surface‐aerated bioreactors are mainly affected by the size of the volumetric oxygen transfer area (a) and the volumetric power input (P∕V_L) that both result from the liquid distribution during shaking. Since Computational Fluid Dynamics (CFD)—commonly applied to simulate the liquid distribution in such bioreactors—needs high computing power, this technique is poorly suited to investigate the influence of many different operating conditions in various scales. Thus, the aim of this paper is to introduce a new mathematical model for calculating the values of a and P∕V_L for liquids with water‐like viscosities. The model equations were derived from the balance of centrifugal and gravitational forces exerted during shaking. A good agreement was found among calculated values for a and P∕V_L, CFD simulation values and empirical results. The newly proposed model enables a time efficient way to calculate the oxygen transfer areas and power input for various shaking frequencies, filling volumes and shaking and reactor diameters. All these parameters can be calculated fast and with little computing power. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1441–1456, 2014     

Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k_La) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k_La) in rocking T‐flasks. We also carried out a scale‐down study at matched k_La between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k_La) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305. © 2012 Wiley Periodicals, Inc.     

Optimization of lipase production by <Emphasis Type="Italic">Staphylococcus warneri</Emphasis> EX17 using the polydimethylsiloxanes artificial oxygen carriers

In this research, the combined effects of polydimethylsiloxane (PDMS) and different conditions of oxygen volumetric mass transfer coefficient (k_La) on lipase production by Staphylococcus warneri EX17 were studied and optimized in bioreactor cultures. Raw glycerol from biodiesel synthesis was used as the sole carbon source. Full-factorial central composite design and the response surface methodology were employed for the experimental design and analysis of the results. The optimal polydimethylsiloxane concentration and mass coefficient transfer (k_La) were found to be 13.5% (v/v) and 181 h⁻¹, respectively. Under these conditions, the maximal cell production obtained was 10.0 g/l, and the volumetric lipase activities of approximately 490 U/l, after 6 h of cultivation. These results are in close agreement with the model predictions. Results obtained in this work reveal the positive effects of PDMS on oxygen volumetric mass transfer coefficient (k_La) in the Staphylococcus warneri EX17 cultivation and lipase production.     

Hydrodynamic and Mass Transfer Characteristics of Three-Phase Gaslift Bioreactor Systems

ABSTRACT:?

This review focuses on the hydrodynamic and mass transfer characteristics of various three-phase, gaslift fluidized bioreactors. The factors affecting the mixing and volumetric mass transfer coefficient (k_La), such as liquid properties, solid particle properties, liquid circulation velocity, superficial gas velocity, bioreactor geometry, are reviewed and discussed. Measurement methods, modeling and empirical correlations are reviewed and compared. To the authors' knowledge, there is no 'generalized' correlation to calculate the volumetric mass transfer coefficient, instead, only 'type-specific' correlations are available in the literature. This is due to the difficulty in modeling the gaslift bioreactor, caused by the variation in geometry, fluid dynamics, and phase interactions. The most important design parameters reported in the literature are: gas hold-up, liquid circulation velocity, 'true' superficial gas velocity, mixing, shear rate, aeration rate and volumetric mass transfer coefficient, k_La.     

A micro-jet array for economic intensification of gas transfer in bioreactors

Bioreactors are of interest for gas-to-liquid conversion of stranded or waste industrial gases, such as CO, CH₄, or syngas. Process economics requires reduction of bioreactor cost and size while maintaining intense production via rapid delivery of gases to the liquid phase (i.e., high k_La). Here, we show a novel bioreactor design that outperforms all known technology in terms of gas transfer energy efficiency (k_La per power density) while operating at high k_La (i.e., near 0.8 s⁻¹). The reactor design uses a micro-jet array to break feedstock gas into a downward microbubble flow. Hydrodynamic and surfactant measurements show the reactor's advanced performance arises from its bubble breakage mechanism, which limits fluid shear to a thin plane located at an optimal location for bubble breakage. Power dissipation and k_L are shown to scale with micro-jet diameter rather than reactor diameter, and the micro-jet array achieves improved performance compared to classical impinging-jets, ejector, or U-loop reactors. The hydrodynamic mechanism by which the micro-jets break bubbles apart is shown to be shearing the bubbles into filaments then fragmentation by surface tension rather than “cutting in half” of bubbles. Guided by these hydrodynamic insights, strategies for industrial design are given. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2710, 2019     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.     

Computational fluid dynamics analysis of mixing and gas–liquid mass transfer in wave bag bioreactor

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (k_La) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors.