Determination of a scale-up factor from mixing time studies in orbitally shaken bioreactors

Efficient mixing in bioreactors is essential in order to avoid concentration gradients which can be harmful for mammalian cells. To study mixing and its scalability in orbitally shaken cylindrical bioreactors, we measured mixing times in containers with nominal volumes from 2 to 1500 L with a colorimetric method using two pH indicators. Four operating parameters were tested: the liquid height, the shaking diameter, the agitation rate, and the inner diameter of the container. The mixing time decreased as the agitation rate increased until a minimal value was reached. As the shaking diameter was reduced, a higher agitation rate was needed to reach the minimal mixing time. The liquid height did not have a significant effect on the mixing time, but for a constant volume, an increase of the inner diameter slightly reduced the mixing time. The fastest mixed zones were close to the wall of the container while the zone in the center of the bulk liquid was the last to achieve homogeneity. Our study showed that the free-surface shape correlated with the mixing regime and that by keeping the inner-to-shaking diameter ratio as well as the Froude number (Fr) constant, the free-surface shapes and the mixing regimes of a 1500-L bioreactor could be mimicked in a 30-L bioreactor. We concluded that the mixing in orbitally shaken cylindrical bioreactors ensures homogeneity for mammalian cell cultures at scales up to 1500 L and that the inner-to-shaking diameter is a suitable scale-up factor for mixing.     

Characterization of gas-liquid mass transfer phenomena in microtiter plates

Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells. The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors. The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L). An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells.     

Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture

The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration. The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained. When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.     

Engineering characterisation of a single well from 24-well and 96-well microtitre plates

The detailed engineering characterisation of shaken microtitre-plate bioreactors will enhance our understanding of microbial and mammalian cell culture in these geometries and will provide guidance on the scale-up of microwell results to laboratory and pilot scale stirred bioreactors. In this work computational fluid dynamics (CFD) is employed to provide a detailed characterisation of fluid mixing, energy dissipation rate and mass transfer in single well bioreactors from deep square 24-well and 96-well microtitre plates. The numerical predictions are generally found to be in good agreement with experimental observation of the fluid motion and measured values of the key engineering parameters. The CFD simulations have shown that liquid mixing is more intensive in 96-well than in 24-well bioreactors due to a significant axial component to the fluid velocity. Liquid motion is strongly dependent on the orbital shaking amplitude which generally has a greater impact than the shaking frequency. Average power consumptions of 70–100 W m⁻³ and 500–1000 W m⁻³, and overall mass transfer coefficient, k_La, values of 0.005–0.028 s⁻¹ and 0.056–0.10 s⁻¹ were obtained for 24-well and 96-well bioreactors respectively at an orbital shaking amplitude of 3 mm and shaking frequencies ranging from 500 rpm to 1500 rpm. The distribution of energy dissipation rates within each bioreactor showed these to be greatest at the walls of the well for both geometries. Batch culture kinetics of E. coli DH5 showed similar maximum specific growth rates and final biomass yields in shaken 24-well and shake flask bioreactors and in stirred miniature and 20 L bioreactors at matched k_La values. The CFD simulations thus give new insights into the local and overall engineering properties of microwell bioreactor geometries and further support their use as high throughput tools for the study and optimisation of microbial and mammalian cell culture kinetics at this scale.     

Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth

Oxygen limitation is one of the most frequent problems associated with the application of shaking bioreactors. The gas-liquid oxygen transfer properties of shaken 48-well microtiter plates (MTPs) were analyzed at different filling volumes, shaking diameters, and shaking frequencies. On the one hand, an optical method based on sulfite oxidation was used as a chemical model system to determine the maximum oxygen transfer capacity (OTR(max)). On the other hand, the Respiration Activity Monitoring System (RAMOS) was applied for online measurement of the oxygen transfer rate (OTR) during growth of the methylotropic yeast Hansenula polymorpha. A proportionality constant between the OTR(max) of the biological system and the OTR(max) of the chemical system were indicated from these data, offering the possibility to transform the whole set of chemical data to biologically relevant conditions. The results exposed "out of phase" shaking conditions at a shaking diameter of 1 mm, which were confirmed by theoretical consideration with the phase number (Ph). At larger shaking diameters (2-50 mm) the oxygen transfer rate in MTPs shaken at high frequencies reached values of up to 0.28 mol/L/h, corresponding to a volumetric mass transfer coefficient (k(L)a) of 1,600 1/h. The specific mass transfer area (a) increases exponentially with the shaking frequency up to values of 2,400 1/m. On the contrary, the mass transfer coefficient (k(L)) is constant at a level of about 0.15 m/h over a wide range of shaking frequencies and shaking diameters. However, at high shaking frequencies, when the complete liquid volume forms a thin film on the cylindric wall of the well, the mass transfer coefficient (k(L)) increases linearly to values of up to 0.76 m/h. Essentially, the present investigation demonstrates that the 48-well plate outperforms the 96-well MTP and shake flasks at widely used operating conditions with respect to oxygen supply. The 48-well plates emerge, therefore, as an excellent alternative for microbial cultivation and expression studies combining the advantages of both the high-throughput 96-well MTP and the classical shaken Erlenmeyer flask.     

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 μg/mL compared with ca. 118 and 990 μg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.     

Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

Oxygen transfer rates in a mammalian cell culture bioreactor equipped with a cell-lift impeller

Measurements of k(L)a were carried out in 1. 5- and 5-L New Brunswick Scientific CelliGen(R) bioreactors. The measured k(L)a in water were identical for both vessel sizes operated in similar condition. The mass transfer rate increased with temperature, mixing speed, and aeration rate, with this last parameter being the most significant. Surface aeration alone gave k(L)a values of 0. 4 to 1. 6 h(-1). A 25% decrease in k(L)a was observed above an aeration rate of 1. 6 vvm. This was caused by the particular foam breaker of the CelliGen bioreactor. Measurements of k(L)a using a mammalian cell culture medium supplemented with 5% fetal calf serum (FCS) have confirmed the negative effect of the foam breaker on k(L)a The measured value in this medium was 1. 2 h(-1) for all aeration rates, more than 60% of which was attributed to surface aeration.     

Oxygen transfer in broths of plant cells at high density

The rheological properties of the culture broths of some plant cells (Cudrania tricuspidata, Vinca rosea, and Agrostemma githago) at high density (10-18 g dry wt/L) were measured, and oxygen transfer in the broths in various bioreactors was investigated. The rheological properties of the broths were dependent on the size, specific gravity, and concentration of the cell aggregates contained in the broths. The broths were non-Newtonian and pseudoplastic fluids. The flow behavior index n was fairly constant (0.53) and the consistency index K varied in proportion to the sixth-to-seventh power of the cell mass concentration M. The apparent viscosity mu(a) of the broths was in proportion to the 6.5th power of M. The oxygen transfer in the broths was discussed on the basis of the results obtained for suspensions of granulated agars (agar concentration, 5.8%) in water, which were similar to the broths in rheological properties. The volumetric oxygen transfer coefficient k(L)a in the broths was dependent on mu(a)(k(L)a proportional, variant mu(a) (-m)) and decreased greatly at a certain apparent viscosity, mu(ac). The values of m and mu(ac) were closely related to the aeration-agitation mechanisms of the bioreactors. The values of mu(ac) in aeration-agitation type bioreactors was larger than that in aeration-type bioreactors, whereas for m, the reverse was true.     

Optimization of oxygen mass transfer in a multiphase bioreactor with perfluorodecalin as a second liquid phase

Oxygenation is an important parameter involved in the design and operation of mixing-sparging bioreactors and it can be analyzed by means of the oxygen mass transfer coefficient (k(L)a). The operational conditions of a stirred, submerged aerated 2-L bioreactor have been optimized by studying the influence of a second liquid phase with higher oxygen affinity (perfluorodecalin or olive oil) in the k(L)a. Using k(L)a measurements, the influence of the following parameters on the oxygen transfer rate was evaluated: the volume of working medium, the type of impellers and their position, the organic phase concentration, the aqueous phase composition, and the concentration of inactive biomass. This study shows that the best experimental conditions were achieved with a perfluorodecalin volume fraction of 0.20, mixing using two Rushton turbines with six vertical blades and in the presence of YPD medium as the aqueous phase, with a k(L)a value of 64.6 h(-1). The addition of 20% of perfluorodecalin in these conditions provided a k(L)a enhancement of 25% when pure water was the aqueous phase and a 230% enhancement when YPD medium was used in comparison to their respective controls (no perfluorodecalin). Furthermore it is shown that the presence of olive oil as a second liquid phase is not beneficial to the oxygen transfer rate enhancement, leading to a decrease in the k(L)a values for all the concentrations studied. It was also observed that the magnitude of the enhancement of the k(L)a values by perfluorodecalin depends on the biomass concentration present.     

Characterization of oxygen transfer in miniature and lab-scale bubble column bioreactors and comparison of microbial growth performance based on constant k(L)a

This work describes the engineering characterization of miniature (2 mL) and laboratory-scale (100 mL) bubble column bioreactors useful for the cultivation of microbial cells. These bioreactors were constructed of glass and used a range of sintered glass gas diffusers with differently sized pores to disperse humidified air within the liquid biomedium. The effect of the pressure of this supplied air on the breakthrough point for gas diffusers with different pore sizes was examined and could be predicted using the Laplace-Young equation. The influence of the superficial gas velocity (u(g)) on the volumetric mass transfer coefficient (k(L)a) was determined, and values of up to 0.09 s(-1) were observed in this work. Two modeling approaches were considered in order to predict and provide comparison criteria. The first related the volumetric power consumption (P/V) to the k(L)a and a good correlation was obtained for differently sized reactors with a given pore size, but this correlation was not satisfactory for bubble columns with different gas diffusers. Values for P/V ranged from about 10 to 400 W.m(-3). Second, a model was developed predicting bubble size (d(b)), bubble rising velocity (u(b)), gas hold-up (phi), liquid side mass transfer coefficient (k(L)), and thus the k(L)a using established theory and empirical correlations. Good agreement was found with our experimental data at different scales and pore sizes. Values for d(b) varied from 0.1 to 0.6 mm, and k(L) values between 1.7 and 9.8 x 10(-4) m.s(-1) were determined. Several E. coli cultivations were performed in the miniature bubble column at low and high k(L)a values, and the results were compared to those from a conventional stirred tank operated under identical k(L)a values. Results from the two systems were similar in terms of biomass growth rate and carbon source utilization.     

Bioactive metabolite production and stress-related hormones in Devil’s claw cell suspension cultures grown in bioreactors

In a previous report, we showed that cell cultures of Harpagophytum procumbens, a South African plant with high medicinal value, accumulate high amounts of anti-inflammatory phenylethanoid glycosides during cultivation in shake-flasks. The aim of the present study was to transfer the phenylethanoid biosynthetic process to a 3-L stirred tank reactor and a 1-L glass-column bioreactor (operated with pulsed aeration). We found that, with stepwise increases in aeration, the stirred tank reactor yielded similar productivities of verbascoside (the major phenylethanoid glycoside in the cells) to those reported for shake-flask cultures (55.68 vs. 54.78 mg verbascoside/L/day, respectively). Transfer in the pulse-aerated column reactor resulted in 165.42 mg verbascoside/L/day, one of the highest yields reported to date. Further, to evaluate the physiological status of the suspended cells in the bioreactors cultures, we examined their hormone levels and compared them to those of cells in shake-flask cultures. While indole-3-acetic acid levels did not differ significantly between the bioreactor and shake-flask cultures, there were considerable differences in their levels of abscisic, jasmonic, and salicylic acids. These results are discussed with respect to relative stress levels in the different cultivation systems.     

Scale-up from shake flasks to pilot-scale production of the plant growth-promoting bacterium Azospirillum brasilense for preparing a liquid inoculant formulation

Azospirillum brasilense has industrial significance as a growth promoter in plants of commercial interest. However, there is no report in the literature disclosing a liquid product produced in pilot-scale bioreactors and is able to be stored at room temperature for more than 2 years. The aim of this work was to scale up a process from a shake flask to a 10-L lab-scale and 1,000-L pilot-scale bioreactor for the production of plant growth-promoting bacterium A. brasilense for a liquid inoculant formulation. Furthermore, this work aimed to determine the shelf life of the liquid formulation stored at room temperature and to increase maize crops yield in greenhouses. Under a constant oxygen mass transfer coefficient (K _L a), a fermentation process was successfully scaled up from shake flasks to 10- and 1,000-L bioreactors. A concentration ranging from 3.5 to 7.5?×?10⁸ CFU/mL was obtained in shake flasks and bioreactors, and after 2 years stored at room temperature, the liquid formulation showed one order of magnitude decrease. Applications of the cultured bacteria in maize yields resulted in increases of up to 95 % in corncobs and 70 % in aboveground biomass.     

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

k(L)a as a predictor for successful probe-independent mammalian cell bioprocesses in orbitally shaken bioreactors

The aim of this study was to gain a better understanding of orbitally shaken bioreactors (OSRs) operated without controllers for pH and dissolved oxygen (DO) concentration. We used cylindrical OSRs with working volumes ranging from 250mL to 200L to determine that the volumetric mass transfer coefficient of oxygen (k(L)a) is a good predictor of the performance of OSRs at different scales. We showed that k(L)a values of 7-10hour(-1) were required to avoid DO limitations and to prevent conditions of low pH during the cultivation of CHO cells. Overall, cell cultures in probe-independent OSRs of different nominal volumes ranging from 250mL to 200L achieved similar cell densities, recombinant protein concentrations, and pH and DO profiles when having the same k(L)a. We conclude that k(L)a is a key parameter for probe-independent bioprocesses in OSRs and can be used as a scale-up factor for their operation.     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

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     

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.     

Scale-up of breast cancer stem cell aggregate cultures to suspension bioreactors

It has been hypothesized that breast tumor formation results from the activity of a scarce population of cells known as Breast Cancer Stem Cells (BrCSCs) and that the development of effective breast cancer therapies may therefore ultimately rely upon the ability to effectively target these cells for eradication. The scarcity of BrCSCs in vivo severely compromises research on these populations, as analyses are restricted to those requiring small cell numbers, and has become a major impediment to the development of therapeutic strategies against breast cancer. Through the culture of murine tissue aggregates containing a population of BrCSCs, this study demonstrates the ability of propagating this scarce population in a controlled and reproducible manner, within suspension bioreactors. A rigorous theoretical framework has been developed in order to understand and characterize the implications of oxygen mass transfer within aggregates upon scale-up and thereby provide a foundation for the scale-up of aggregate cultures. A two-factor, two-level factorial experimental design was also performed in order to assess the effects of inoculation density and hydrodynamic shear upon cell yield. We discovered that the culture of the murine aggregates in a relatively low shear environment (tau(max) = 0.20 Pa) and inoculated at 3.50 x 10(4) cells/mL resulted in the best yields for the range of conditions investigated in suspension bioreactors. A detailed study on the oxygen uptake kinetics of the aggregates also revealed that the uptake rates were not significantly affected by mass transfer limitations, as uptake rates of aggregate cultures were found to be comparable to those observed in single cell cultures. Cells propagated in a process controlled 500 mL suspension bioreactor resulted in growth kinetics that were comparable to those observed in 125 mL bioreactors. Doubling times in the 500 mL vessel were found to be 23.9 h and attained a maximum cell density of 1.20 x 10(6) cells/mL. After enumerating the number of BrCSCs, this resulted in an approximately 20-fold increase in BrCSC numbers in batch suspension cultures. With greater attention being applied to BrCSCs, their propagation in suspension bioreactors makes available experimental avenues that are not currently accessible and may thereby enable the development of more effective therapeutic drugs for the treatment of breast cancer.