Sparged Bioreactors Research Articles

Modelling of Bubble Breakage and Coalescence in Stirred and Sparged Bioreactor Using the Euler-Lagrange Approach

Modelling of Bubble Breakage and Coalescence in Stirred and Sparged Bioreactor Using the Euler-Lagrange Approach

Investigation of interfacial properties of pure and mixed poloxamers for surfactant-mediated shear protection of mammalian cells

The Poloxamer family of surfactants are commonly used in the biopharmaceutical industry as cell culture media additives to protect cells from the turbulent environment of sparged bioreactors. Despite the widespread use of poloxamers in cell culture, their performance as cell protectants varies depending on their physical structure, molecular weight, and batch-to-batch composition. In this study, the interfacial properties of Poloxamer 188 (P188), Poloxamer 407 (P407), and a mixture of P188 and P407 were characterized to investigate the mechanism of surfactant-mediated shear protection of mammalian cells. The foam stability and equilibrium surface tension of these surfactant systems correlated with their ability to mitigate physical damage to cells in a turbulent environment. We demonstrate that while P188 can function as highly effective shear protectant, the presence of a surface-active contaminant can greatly hinder its protective characteristics. P407 was found to function as such an interfacially active “impurity,” disrupting shear protection when mixed with P188 by preferentially adsorbing to the gas-liquid and membrane-liquid interface. Addition of surface-active impurities altered the interfacial properties of the surfactant system and could be detected using an equilibrium surface tension assay. The mechanism of disruption by P407 was determined to be independent of cell-to-bubble attachment, suggesting that poloxamer adsorption to and subsequent reinforcement of the cell membrane may play a key role in protecting cells in high shear environments. This investigation contributes to our understanding of the mechanism of surfactant-mediated shear protection of cells and demonstrates that a surface tension assay can be utilized as a screening tool to ensure that poloxamer lots are free of surface active impurities.

Energy consumption due to mixing and mass transfer in a wave photobioreactor

Photobioreactors for algal cultivation commonly use energy intensive gas sparging to facilitate mixing and gas-liquid mass transfer. A wave reactor provides an alternative to sparging by providing surface aeration and mixing by means of a rocking platform. Wave bioreactors have been used previously for microbial cultivation but this is the first report to assess microalgal cultivation in a wave reactor with respect to the energy consumed for mixing and mass transfer. Tracer studies and the dynamic gassing-in method revealed mixing times (18–71s) and kLaCO2 (9–66h−1) comparable to previously reported values for wave reactors. The specific power input for the wave reactor, measured using calorimetry, ranged from 57.6 to 903.3Wm−3 at rocking rates from 15 to 40rpm, at a 10° rocking angle. Scenedesmus sp. was cultivated in the wave reactor at 25rpm and 10°, 15rpm and 8°, as well as 15rpm and 4° (kLaCO2 9–38h−1) all at a light intensity of 140μmolm−2s−1. A maximum biomass concentration of 2.25gL−1 and overall biomass productivity of 0.187gL−1d−1 were obtained at 25rpm and 10°. There was no significant variation in these values across the rocking conditions investigated, allowing energy reduction without adverse effects to microalgal cultivation. The successful cultivation of Scenedesmus sp. in the wave reactor indicated the potential for these well-mixed, surface aerated reactors in microalgal applications as a promising alternative to sparged bioreactors.

Cultivation of Chlorella Vulgaris Using Airlift Photobioreactor Sparged with 5%CO 2 -Air as a Biofixing Process

The present paper addresses cultivation of Chlorella vulgaris microalgae using airlift photobioreactor that sparged with 5% CO 2 /air. The experimental data were compared with that obtained from bioreactor aerated with air and unsparged bioreactor. The results showed that the concentration of biomass is 0.36 g l -1 in sparged bioreactor with CO2/air, while, the concentration of biomass reached to 0.069 g l -1 in the unsparged bioreactor. They showed also that aerated ioreactor.with CO2/air gives more biomass production even the bioreactor was aerated with air. This study proved that application of sparging system for ultivation of Chlorella vulgaris microalgae using either CO2/air mixture or air has a significant growth rate, since the bioreactors become more thermodynamically favorable and provide impetus for a higher level of production. biofixing process

Free surface oxygen transfer in large aspect ratio unbaffled bio-reactors, with or without draft-tube

It is widely accepted that animal cell damage in aerated bioreactors is mainly related to the bursting of bubbles at the air–liquid interface. A viable alternative to sparged bioreactors may be represented by uncovered unbaffled stirred tanks, which have been recently found to be able to provide sufficient mass transfer through the deep free surface vortex which takes place under agitation conditions. As a matter of fact, if the vortex is not allowed to reach impeller blades, no bubble formation and subsequent bursting at the free-surface, along with relevant cells damage, occurs.In this work oxygen transfer performance of large aspect ratio unbaffled stirred bioreactors, either equipped or not with an internal draft tube, is presented, in view of their use as biochemical reactors especially suited for shear sensitive cell cultivation.

Investigation of low viability in sparged bioreactor CHO cell cultures points to variability in the Pluronic F-68 shear protecting component of cell culture media

An unexpected 6–9% drop of viability in suspension CHO cell cultures 24–48h post-inoculation in sparged bioreactors, paired with reduced overall cell growth by an average of 43%, occurred upon switching to new serum and hydrolysate-free chemically defined media (CDM) lots. An investigation was undertaken to identify sources of variability. First, chemical/biological contaminants, including cleaning procedures, gas source/quality, compromised bioreactor parts and mycoplasma contamination, were ruled out. Second, we tested a gradient of sparging rates and observed that early post-inoculation cell death increased with the gas flow rate. While bubbles are known to cause cell damage, this effect was not previously observed at the high sparging rate used. Third, because hydrodynamic damage appeared to be responsible for cell death, we tested the addition of increased levels of surfactant Pluronic F-68 (PF-68), which reduces cell-to-bubble attachment and lowers dynamic surface tension, to the new CDM lots. This restored the viable cell density and viability profiles. Measurement of the dynamic surface tension of the CDM lots employed in this study indicated variability up to 8% between CDM lots supplemented with different PF-68 lots. In summary, our observations suggest that the raw material quality of PF-68 lots can alter the ability to lower the surface tension in chemically-defined cell culture media and significantly impact performance of CHO cell cultures in sparged bioreactors.

Effects of bubble–liquid two‐phase turbulent hydrodynamics on cell damage in sparged bioreactor

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas–liquid bioreactors have not been performed to date. In this work, a second-order moment (SOM) bubble–liquid two-phase turbulent model based on the two-fluid continuum approach is used to investigate the gas–liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble–liquid two-phase fluctuation velocities correlation transport equations, the anisotropy of two-phase stresses and the bubble– liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress-induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region.

Impact of aeration strategy on CHO cell performance during antibody production

Stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. The hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. Here we investigate the sub-lethal effects of different aeration strategies on Chinese hamster ovary cells during monoclonal antibody production. Two gas delivery systems were employed to study the presence and absence of the air-liquid interface: bubbled direct gas sparging and a non-bubbled diffusive silicone membrane system. Additionally, the effect of higher gas flow rate in the sparged bioreactor was examined. Both aeration systems were run using chemically defined media with and without the shear protectant Pluronic F-68 (PF-68). Cells were unable to grow with direct gas sparging without PF-68; however, when a silicone membrane aeration system was implemented growth was comparable to the sparged bioreactor with PF-68, indicating the necessity of shear protectants in the presence of bubbles. The cultures exposed to increased hydrodynamic stress were shown by flow cytometry to have decreased F-actin intensity within the cytoskeleton and enter apoptosis earlier. This indicates that these conditions elicit a sub-lethal physiological change in cells that would not be detected by the at-line assays which are normally implemented during cell culture. These physiological changes only result in a difference in continuous centrifugation performance under high flow rate conditions. Product quality was more strongly affected by culture age than the hydrodynamic conditions tested.

Production of yellow fever virus in microcarrier-based Vero cell cultures

In this work, the propagation of the 17DD yellow fever virus in Vero cells grown on Cytodex-1 microcarriers was evaluated. After verifying that upon infection the virus adsorption step could be performed under continuous agitation, experiments were carried out in spinners and sparged lab-scale stirred-tank bioreactor to evaluate the use of a commercial serum-free medium (VP-SFM) and to investigate the effects of multiplicity of infection (MOI) and time of infection (TOI) on virus production. Virus titers as high as 8.4 × 10 8 pfu/mL were obtained upon infection with MOI of 0.02 and TOI of 3 days, using the serum-free medium in the sparged bioreactor.

NS0 cell damage by high gas velocity sparging in protein‐free and cholesterol‐free cultures

Recent developments in high cell density and high productivity fed-batch animal cell cultures have placed a high demand on oxygenation and carbon dioxide removal in bioreactors. The high oxygen demand is often met by increasing agitation and sparging rates of air/O2 in the bioreactors. However, as we demonstrate in this study, an increase of gas sparging can result in cell damage at the sparger site due to high gas entrance velocities. Previous studies have showed that gas bubble breakup at the culture surface was primarily responsible for cell damage in sparged bioreactors. Such cell damage can be reduced by use of surfactants such as Pluronic F-68 in the culture. In our results, where NS0 cells were grown in a protein-free and cholesterol-free medium containing 0.5 g/L Pluronic F-68, high gas entrance velocity at the sparger site was observed as the second mechanism for cell damage. Experiments were performed in scaled-down spinners to model the effect of hydrodynamic force resulting from high gas velocities on antibody-producing NS0 cells. Cell growth and cell death were described by first-order kinetics. Cell death rate constant increased significantly from 0.04 to 0.18 day(-1) with increasing gas entrance velocity from 2.3 to 82.9 m/s at the sparger site. The critical gas entrance velocity for the NS0 cell line studied was found to be approximately 30 m/s; velocities greater than 30 m/s caused cell damage which resulted in reduced viability and consequently reduced antibody production. Observations from a second cholesterol-independent NS0 cell line confirmed the occurrence of cell damage due to high gas velocities. Increasing the concentration of Pluronic F-68 from 0.5 to 2 g/L had no additional protective effect on cell damage associated with high gas velocity at the sparger. The results of gas velocity analysis for cell damage have been applied in two case studies of large-scale antibody manufacturing. The first is a troubleshooting study for antibody production carried out in a 600 L bioreactor, and the second is the development of a gas sparger design for a large bioreactor scale (e.g., 10,000 L) for antibody manufacturing.

An investigation of small‐molecule surfactants to potentially replace pluronic F‐68 for reducing bubble‐associated cell damage

It is well known that bubble rupture has a detrimental effect on mammalian cells. As a result, Pluronic F-68 (PF-68), a nonionic surfactant, is commonly used to reduce bubble-associated cell damage in sparged bioreactors. While PF-68 is currently effective, there is a concern with respect to its decrease in effectiveness as cell concentrations increase (Ma et al., 2004, Biotechnol Prog 20:1183-1191). In addition, having more than one effective surfactant for cell culture is also highly desirable. Given the empirical nature in which PF-68 was initially discovered as a cell culture additive, a structure-performance study of small molecule surfactants, a distinct group which have been previously investigated for other purposes, was performed in an attempt to find a replacement for PF-68. In this study, a generic platform was established to initially screen both the type and concentration of these surfactants for cytotoxicity. Promising candidates where then evaluated for their ability to rapidly lower the surface tension (dynamic surface tension) of culture media and their ability to prevent cell-bubble attachment in a specially developed bubble creation and collection system. Several promising small- molecule surfactants, and their effective concentration, were identified, which can reduce cell-bubble attachment efficiently without being harmful to cells.

The Sensitivity of the Dinoflagellate Crypthecodinium cohnii to Transient Hydrodynamic Forces and Cell‐Bubble Interactions

The increased interest in the benefits of omega-3 fatty acids for human health has resulted in the commercial development of the dinoflagellate Crypthecodinium cohnii for production of docosahexaenoic acid (DHA). The growing market demand for DHA requires highly efficient, very large scale cultures of DHA. While the effects of hydrodynamic forces on dinoflagellates have been investigated for several decades, the majority of the work focused on the negative effects of oceanic turbulence on the population growth of environmentally important dinoflagellates. In contrast, significantly less is known on the effect of hydrodynamic forces encountered by algae in bioprocesses. Unlike other studies conducted on algae, this study employed a microfluidic, flow contraction device to evaluate the effect of transient hydrodynamic forces on C. cohnii cells. It was found that C. cohnii cells can sustain the energy dissipation rate of 5.8 x 10(7) W/m3 without lysis. However, an obvious sublethal effect, the loss of flagella, was observed at a lower level of 1.6 x 10(7) W/m3. Finally the cell-bubble interaction and the effect of bubble rupture were also explored to simulate the conditions of sparged bioreactors.

Characterization of the localized hydrodynamic shear forces and dissolved oxygen distribution in sparged bioreactors

Detailed, high-resolution numerical simulations of the bubbly flows, used for oxygen delivery and mixing in mammalian cell suspensions, have been performed. The hydrodynamics, shear and normal forces, mass transfer and mass transport from and around individual bubbles and bubble clusters were resolved for different operating conditions, that is, Weber, Morton, and Schmidt numbers. Suspended animal (e.g., mammalian, insect) cells are known to be susceptible to damage potentially leading to cell death, caused by hydrodynamic stresses and oxygen deprivation. Better knowledge of the magnitude of the shear forces and the extent of mixing of the dissolved oxygen in sparged bioreactors can have a significant impact on their future design and optimization. Therefore, the computed liquid-phase velocity fields were used to calculate and compare the local shear in different types of single bubble wakes and in bubble clusters. Oxygen mass transfer and dissolved oxygen transport were resolved to examine oxygen supply to the cells in the different types of flows.

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.

Sparging and agitation-induced injury of cultured animals cells: Do cell-to-bubble interactions in the bulk liquid injure cells?

It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. (c) 1996 John Wiley & Sons, Inc.

Oxygen gradients in small and big sparged insect-cell bioreactors

It should be clear from the above that the calculations described here are at best rough estimations yielding order-of-magnitude values. Even though, the following general conclusions can be drawn. The gradients in stagnant layers surrounding the particles which are characteristic for animal-cell bioreactors are relatively small as compared to the gradients which can be expected in the bulk-liquid phases of the three bioreactors considered, in particular to the gradients in the stagnant layer surrounding the air bubbles. It can be concluded that under almost all circumstances gradients are likely to exist and can be very steep in larger vessels and in particular at high cell densities. The effects of gradients, however, are largely unknown; therefore research on the effects of gradients on specific and volumetric productivities and product quality seems to be an interesting area.

Analysis of cell‐to‐bubble attachment in sparged bioreactors in the presence of cell‐protecting additives

To investigate the mechanisms of cell protection provided by medium additives against animal cell injury in sparged bioreactors, we have analyzed the effect of various additives on the cell-to-bubble attachment process using CHO cells in suspension. Cell-to-bubble attachment was examined using three experimental techniques: (1) cell-bubble induction time analysis (cell-to-bubble attachment times); (2) forming thin liquid films and observing the movement and location of cells in the thin films; and (3) foam flotation experiments. The induction times we measured for the various additives are as follows: no additive (50 to 500 ms), polyvinyl pyrrolidone (PVP: 20 to 500 ms), polyethylene glycol (PEG: 200 to 1000 ms), 3% serum (500 to 1000 ms), polyvinyl alcohol (PVA: 2 to 10 s), Pluronic F68 (5 to 20 s), and Methocel (20 to 60 s). In the thin film formation experiments, cells in medium with either F68, PVA, or Methocel quickly flowed out of draining thin liquid films and entered the plateau border. When using media with no additive or with serum, the flow of cells out of the thin liquid film and film drainage were slower than for media containing Pluronic F68. PVA, or Methocel. With PVP and PEG, the thin film drainage was much slower and cells remained trapped in the film. For the foam flotation experiments, a separation factor (ratio of cell concentration in the foam catch to that in the bubble column) was determined for the various additives. In the order of increasing separation factors (i.e., increasing cell attachment to bubbles), the additives are as follows: Methocel, PVA, Pluronic F68, 3% serum, serum-free medium with no additives, PEG, and PVP. Based on the results of these three different cell-to-bubble attachment experiments, we have classified the cell-protecting additives into three groups: (1) Pluronic F68, PVA, and Methocel (reduced cell-to-bubble attachment); (2) PEG and PVP (high or increased cell-to-bubble attachment); and (3) FBS (reduced cell attachment butslower drainage films compared with F68, PVA, and Methocel with some cell entrapment in those films). These phenomena are discussed in relation to the interfacial properties of the media reported in a companion Study (this issue). (c) 1995 John Wiley & Sons Inc.

Unified modeling framework of cell death due to bubbles in agitated and sparged bioreactors

A modeling framework is proposed to assess the detrimental effects of air sparging and other bubble phenomena (vortex entrainment, coalescence, bursting) on freely suspended cells in an aerated, agitated bioreactor. It is assumed that cells may be rendered nonviable by bubble breakup/coalescence within the medium, by bubble formation at the sparger, or by bubble bursting at the free surface. Some plausible mechanisms are argued from the energetic view point. The dominant parameters in each case are the cell-bubble encounter rate and the bubble breakup/bursting rate. These inactivation processes lead to a Michaelis-Menten expression for the specific cell death rate, which is shown to be linearly proportional to the specific bubble interfacial area (total bubble surface area per unit volume of media). By using published viable cell concentration data for retarded growth of mammalian cells due to sparging, the interfacial area correlation is demonstrated. The method is generalized to aerated bioreactor conditions. The article offers a unique, consistent perspective on how cell death can be viewed.

Cells and bubbles in sparged bioreactors

Ever since animal cells have been grown in-vitro, various techniques have been used to supply the cells with oxygen. The most simple and commonly used 'large-scale' technique to provide oxygen is through the introduction of gas bubbles. However, almost since the beginning of in-vitro cell culture, empirical observations have indicated that bubbles can be detrimental to the cells. This review will discuss the background of the problem, review the relevant research on the topic, attempt to provide a coherent summary of what we know from all of this research, and finally outline what still needs to be investigated. Specific topics to be covered include: experimental correlations of cell damage with bubbles, cell attachment to bubbles, the hydrodynamics of bubble rupture, bioreactor studies, visualization studies, and computer simulations and qualification of cell death as a result of bubble rupture.

AN EXPERIMENTAL STUDY OF BUBBLE FORMATION TRANSITIONS AT SIEVE PLATE SPARGERS

Abstract Evidence has been presented in the literature suggesting that certain interracial phenomena may produce cellular damage in culture operations. In an earlier study of bubble-driven fluid motions in a sparged bioreactor reported by Glasgow et al. (1992), some energetic transitions associated with the bubble formation process were observed at sieve plate spargers. These transitions, involving both the active hole location and mode, were found to be independent of gas supply pressure fluctuations but quite sensitive to gas chamber volume. In the present study, five different experimental techniques have been employed in an effort to better characterize these transitions. Macrovideography and photographic flow visualization were initially used to record, classify, and count the transitions and it was discovered that frequency of transition was a qualitatively reproducible function of superficial gas velocity. A light-scattering experiment was then devised in which fluctuations in received light intens...