Scale-up of biotransformation process in stirred tank reactor using dual impeller bioreactor

The gas-liquid mass transfer coefficient K(L)a in the fermenter is a strong function of mode of energy dissipation and physico-chemical properties of the liquid media. A combination of disc turbine (DT) and pitched blade turbine down flow (PTD) impellers has been tested in laboratory bioreactor for gas hold-up and gas-liquid mass transfer performance for the growth and biotransformation medium for an yeast isolate VS1 capable of biotransforming benzaldehyde to L-phenyl acetyl carbinol (L-PAC) and compared with those in water.Correlations have been developed for the prediction of the fractional gas hold-up and gas-liquid mass transfer coefficient for the above media. The mass transfer coefficient and respiration rate have been determined in the shake flask for the growth as well as for biotransformation medium. These results, then have been used to optimize the operating parameters (impeller speed and aeration) for growth and biotransformation in a laboratory bioreactor. The comparison of cell mass production and L-PAC production in the bioreactor has been done with that obtained in shake flask studies.     

Generation of small bubbles and small bubble-liquid mass transfer in airlift reactors containing highly viscous liquids

The time-dependent gas hold-up is investigated during the aeration of the Saccharomyces cerevisiae suspension, the aqueous saccharose solutions and the glycerol solutions in the external loop airlift reactor. Due to the time-dependent bubble size distribution the fraction of the small bubble hold-up in the total gas hold-up decreases with an increase of the gas flow rate and with a decrease of the viscosity. The course of the accumulation process of the small bubbles is described by the first-order kinetic equation. The small bubble accumulation rate is investigated in the airlift reactor and the bubble column. It is showed that the small bubbles form and disappear exclusively in the riser of the airlift reactor. It is found that the small bubble-liquid mass transfer coefficient is several times larger than the overall oxygen transfer coefficient.     

Characterization of a pilot plant airlift tower loop reactor: III. Evaluation of local properties of the dispersed gas phase during yeast cultivation and in model media

The local properties of the dispersed gas phase (gasholdup, bubble diamater, and bubble velocity) were measured and evaluated at different positions in the riser and downcomer of a pilot plant reactor and, for comparison, in a laboratory reactor. These were described in Parts I and II of this series of articles during yeast cultivation and with model media. In the riser of the pilot plant reactor, the local gas holdup and bubble velocities varied only slightly in axial direction. The gas holdup increased considerably, while the bubble velocity increased only slightly with aeration rate. The bubble size diminished with increasing distance from the aerator in the riser, since the primary bubble size was larger than the equilibrium bubble size. In the downcomer, the mean bubble size was smaller than in the riser. The mean bubble size varied only slightly, the bubble velocity was accelerated, and the gas holdup decreased from top to bottom in the downcomer. In pilot plant at constant aeration rate, the properties of the dispersed phase were nearly constant during the batch cultivation, i.e., they depended only slightly on the cell concentration. In the laboratory reactor, the mean bubble sizes were much larger than in the pilot plant reactor. In the laboratory reactor, the bubble velocities in the riser and downcomer increased, and the mean gas holdup and bubble diameter in the downcomer remained constant as the aeration rate was increased.     

Study of hydrodynamics in microcarrier culture spinner vessels: A particle tracking velocimetry approach

Three-dimensional particle tracking velocimetry (3-D PTV), a modern, quantitative, visualization tool, has been applied to the characterization of the flow field in the impeller region of cell culture reactor vessels. The experimental system used here is a 250-mL microcarrier spinner vessel. The studies were conducted at three different agitation rates, 90, 150, and 210 rpm, corresponding to healthy, mildly damaging, and severely damaging shear intensities, respectively. The flow can be classified into three regions: a predominantly tangential (azimuthal) flow generated by the impeller; a trailing vortex region coming off the impeller tip; and a converging flow region close to the center of the vessel. The latter two are the regions of highest velocity gradients. Energy dissipation rates due to mean velocity gradients were also calculated to characterize the impeller stream. Local specific energy dissipation rates > 10,000 erg/(cm(3)sec) . have been measured. It is proposed that the critical regions for microcarrier culture damage due to impeller hydrodynamics are the trailing vortex region and the high energy converging flow region. Graphical representation of the mean velocity flow fields and the distribution of energy dissipation rates in the impeller region are also presented here. The merits of using the dissipation function (measure of specific energy dissipation rate) as a possible scale-up parameter are also discussed. (c) 1996 John Wiley & Sons, Inc.     

Pilot-scale culture ofCoffea arabica in a novel loop fluidised bed reactor

A novel bubble free loop fluidized bed reactor for plant cell cultures was developed and tested usingCoffea arabica as a model cell line. The effects of main operational parameters like morphology and size of inoculum, oxygen supply as well as recirculation of sparingly soluble gases on cell growth and alkaloid production rates in this reactor were studied and the results were compared with standard shake flask experiments. By on-line monitoring of biomass and oxygen uptake rates the main kinetic parameters for cell growth and alkaloid production were evaluated. It was demonstrated that the novel reactor is easy to run and is particularly adequate for measuring kinetic parameters necessary for scale up.     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

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.     

Local gas hydrodynamics in an external-loop airlift reactor: newtonian and non-newtonian fluids

Measurements of local gas phase characteristics are obtained in an external-loop airlift reactor filled with newtonian or viscous non-newtonian liquids. A double-optical fiber probe technique is used. It allows the determination of the axial and radial profiles of gas hold-up, bubbling frequency, bubble size and velocity. In the case of air-water system, the results show a strong effect of radial liquid velocity variation on the gas flow characteristics at the bottom of the riser. In the case of highly viscous non-newtonian solution, the gas flow is strongly affected by the gas distribution just above the gas sparger. This study also points out the bubble coalescence and the break-up phenomena in different liquids and levels in the reactor. Furthermore, the local measurements of bubble size and velocity allows to gain more detailed information on the dynamics of the bubble-flow and shows a tendency of large bubbles to circulate in the column center.     

A perfusion system for antibody production by shear-sensitive hybridoma cells in a stirred reactor

Summary A shear-sensitive hybridoma cell line, incapable of growth or antibody production in spinner or shake flasks agitated at 40 rpm, was grown successfully in a perfusion propagation system consisting of a bioreactor (1.5 liter), stirred with a cell-lift impeller at 60 rpm, and a tangential flow filtration unit for removal of spent culture medium from the reactor. The culture was maintained over a 48 day period and cell numbers reached 1.8 × 10⁷ cells/ml. Maximal monoclonal antibody concentration was 800 ug/ml, indicating a productivity of 504 mg/day.     

Evaluation of the killing volume of gas bubbles in sparged animal cell culture bioreactors

The detrimental effect of direct gas sparging on insect cells was investigated in bubble columns with various gas flow rates and bubble sizes. The first-order cell death rate was shown to be directly proportional to the gas flow rate and inversely proportional to the bubble size. The specific killing volume of a bubble, killing volume per unit volume of bubble, was found to have a linear correlation with the specific interfacial area of a bubble. Based on these experimental results and the analysis of a bursting bubble at the liquid surface, it was concluded that the killing volume of a bubble is in the liquid layer surrounding the bubble before its rupture, and most important, in the liquid layer beneath the bubble cavity. Cell damage in the bubble film cap was relatively insignificant compared to that in the liquid layer underneath the bubble cavity, except for very large bubbles (i.e., bubble diameter over 5 mm).     

Effects of paddle impeller geometry on power input and mass transfer in small-scale animal cell culture vessels

Process scaleup for stirred-tank animal cell cultures such as suspension and microcarrier cultures often begins at the bench scale in small spinner vessels. In order to initiate process development under the proper conditions, it is essential to know the physical conditions under which the cells are grown. In this article, power inputs and surface oxygen transfer rates to culture medium in 500-mL Corning spinner vessels were determined as a function of the impeller geometry, impeller height, and agitation speed. The results obtained indicate that power dissipation dependency differs from literature correlations and may compromise scale up at constant power input from these vessels. These results are of general utility to researchers using small-scale vessels.     

High oxygen transfer rate in a new aeration system using hollow fiber membrane

A new type of bubble aeration column called a hollow fiber membrane (HFM) aeration column was proposed, which was featured in the use of hollow fiber membranes and gave a high bubble density in the column. The value of k(L)a was increased by modifying the membrane surface for making the pore size smaller. The Sauter mean diameter of bubbles (D(vs)) was 2.0 +/- 0.1 mm in the range of the superficial gas velocity from 0.02 m s(-1) to 0.065 m s(-1), while that obtained for the bubbles near the membrane was 811 mum at the superficial gas velocity of 4.0 x 10(-4) m s(-1). The difference was ascribed to the effect of coalescence of bubbles. The value of K(L)a increased in proportion to the superficial gas velocity up to 0.02 m s(-1), and was almost constant above 0.03 m s(-1). The maximum value of k(L)a, 2.5 s(-1), was higher than those of the other aeration columns reported previously. The pneumatic power consumption per unit liquid volume (P(v)) for obtaining the same k(L)a was the smallest in the HFM aeration columns. P(v), for obtaining the same interfacial area of bubbles per liquid volume, was also lower than those for other types of aeration columns. It was suggested from the measurement of bubble diameter that the larger interfacial area generated in the HFM aeration column ascribes to the larger gas holdup than the smaller D(vs). (c) 1992 John Wiley & Sons, Inc.     

Carbon monoxide mass transfer for syngas fermentation in a stirred tank reactor with dual impeller configurations

This study compares the power demand and gas-liquid volumetric mass transfer coefficient, kLa, in a stirred tank reactor (STR) (T = 0.211 m) using different impeller designs and schemes in a carbon monoxide-water system, which is applicable to synthesis gas (syngas) fermentation. Eleven different impeller schemes were tested over a range of operating conditions typically associated with the "after large cavity" region (ALC) of a Rushton-type turbine (D/T = 0.35). It is found that the dual Rushton-type impeller scheme exhibits the highest volumetric mass transfer rates for all operating conditions; however, it also displays the lowest mass transfer performance (defined as the volumetric mass transfer coefficient per unit power input) for all conditions due to its high power consumption. Dual impeller schemes with an axial flow impeller as the top impeller show improved mass transfer rates without dramatic increases in power draw. At high gas flow rates, dual impeller schemes with a lower concave impeller have kLa values similar to those of the Rushton-type dual impeller schemes but show improved mass transfer performance. It is believed that the mass transfer performance can be further enhanced for the bottom concave impeller schemes by operating at conditions beyond the ALC region defined for Rushton-type impellers because the concave impeller can handle higher gas flow rates prior to flooding.     

The Super-Spinner: A low cost animal cell culture bioreactor for the CO2 incubator

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran^® flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO₂ conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTR_max) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTR_max was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Continuous insect cell (Sf-9) culture with aeration through sparging

The continuous growth of Spodoptera frugiperda Sf-9 cells in a 250-ml blown-glass jacketed spinner flask under a direct air sparging environment was investigated. Even at 220 ml working volume (about 90% of total volume), this spinner flask provided good mixing and oxygenation as demonstrated by a higher cell density compared with fermentor cultures. This eliminates a common limitation of the traditional spinner flask, namely much lower cell density at high working volume. Furthermore, this spinner flask has been run with Sf-9 cell culture at five different dilution rates and two different air sparging rates at steady state, demonstrating its utility in research applications where cell size, metabolic activity and environmental conditions can be constantly maintained. In addition to demonstrating the utility of the reactor, three novel points are made in this report. First, cell density in continuous cultures is increased significantly due to a high agitation rate and, especially, air sparging rate, which is seldom used in animal cell or insect cell culture. Second, there is no apparent difference in the specific death rate at two different sparging rates (0.0093 vvm and 0.0125 vvm). Finally, we have maintained Sf-9 cells for more than 4 months in a continuous culture using a serum-free medium without loss of recombinant protein expression in infected cells.     

Characterization of agitation environments in 250 ml spinner vessel, 3 L,and 20 L reactor vessels used for animal cell microcarrier culture

Three dimensional particle tracking velocimetry (3-D PTV) was used to characterize the flow fields in the impeller region of three microcarrier reactor vessels. Three typical cell culture bioreactors were chosen: 250 ml small-scale spinner vessels, 3 L bench-scale reactor, and 20 L medium-scale reactor. Conditions studied correspond to the actual operating conditions in industrial setting and were determined based on the current scale-up paradigm: the Kolmogorov eddy length criterion. In this paper we present characterization of hydrodynamics on the basis of flow structures produced because of agitation. Flow structures were determined from 3-D mean velocity results obtained using 3-D PTV. Although the impellers used in 3 L and 20 L reactors were almost identical, the flow structures produced in the two reactors differed considerably. Results indicate that near geometric scale up does not necessarily amount to scale-up of flow patterns and indicates that intensity as well as distribution of energy may vary considerably during such a scale-up.     

The tumbling reactor: a new reactor design for biotechnology Cultivation of the phototrophic micro-alga Scenedesmus communis

A new system for the growth of mammalian cells was developed and its biotechnological suitability was proven by the sterile cultivation of the phototrophic micro-alga Scenedesmus communis, as an easy to cultivate model organism. For this purpose, the bioreactor was combined with a newly developed equipment for irradiation of large scale cultures of phototrophic micro-organisms. Cultivation was performed in a 40 liter scale at 25°C and a pH of 6.0 to 6.5. The aeration gas consisted of 2% or 7% of CO2 in N2. The aeration rate was Vgas = 19.6 L.h-1, the stirring speed was n = 70 or 56 min-1. The new reactor consists of 8 totally immersed fluorescent tubes located around a cylinder for bubble free aeration which is moving within the cultivation broth. Results of the cultivation reveal the suitability of this newly designed system for biotechnological use and its high scale-up potential.     

Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda (Sf-9) insect cells.

Spodoptera frugiperda (Sf-9) insect cells have been grown in serum-free medium in 250-ml spinner flasks. The maximum cell density obtained in these cultures was dependent on the aeration rate of the culture. Similar yields of uninfected cells were obtained when cultures were stirred in spinner flasks at 80 rev min-1 and in a 4-1 stirred-tank bioreactor and the dissolved oxygen in the bioreactor was controlled at 20% of air saturation. Cells were infected with a recombinant baculovirus at different multiplicities of infection: the timing and maximum level of expression of the recombinant protein were dependent on the multiplicity of infection, the cell density at infection, and on the aeration rate of the culture. Oxygen-limited growth resulted in undetectable levels of recombinant protein (< 6 ng recombinant protein 10(-7) cells). Compared with the maximum yields observed in spinner flask cultures, higher levels of recombinant protein were produced when cells were grown and infected in the bioreactor. The level of dissolved oxygen in the bioreactor was controlled at 50% of air saturation.     

Long-term Continuous Production of Monoclonal Antibody by Hybridoma Cells Immobilized in a Fibrous-Bed Bioreactor

The kinetics and long-term stability of continuous production of monoclonal antibody IgG2b by hybridoma HD-24 cells immobilized in a fibrous-bed bioreactor (FBB) were studied for a period of ～8 months. The cells were immobilized in the fibrous bed by surface attachment of cells and entrapment of large cell clumps in the void space of the fibrous matrix. A high viable cell density of 1.01 × 10⁸/ml was attained in the bioreactor, which was about 63 times higher than those in conventional T-flask and spinner flask cultures. The continuous FBB produced IgG at a concentration of ～0.5 g/l, with reactor productivity of ～7 mg/h·l, which was about 23 times higher than those from conventional T-flask and spinner flask cultures. The IgG concentration can be further increased to ～0.67 g/l by using higher feed (glucose and glutamine) concentrations and running the reactor at a recycle batch or fed-batch mode. The long-term performance of this bioreactor was also evaluated. For a period of 36 days monitored, the MAb produced in the continuous well-mixed bioreactor at 50 h retention time (0.02/h dilution rate) was maintained at a steady concentration level of ～0.3 g/l with less than 8% drift. At the end of the study, it was found that ～25% of the cells were strongly attached to the fiber surfaces and the other ～75% entrapped or weakly immobilized in the fibrous matrix. The strongly attached cells had a high viability of ～90%, compared to ～75% for cells weakly immobilized and only ～1.4% for freely suspended cells, suggesting that the fibrous matrix preferentially retained and protected the viable (productive) cells. The FBB thus was able to maintain its long-term productivity because nonviable and dead cells were continuously washed off from the fibrous matrix. The high MAb concentration and production rate and excellent stability for continuous long-term production obtained in this study compare favorably to other bioreactor studies reported in the literature. The reactor performance can be further improved by providing better pH and aeration controls at higher feed concentrations. The FBB is easy to operate and scale-up, and thus can be used economically for industrial production of MAb.