[Show abstract][Hide abstract] ABSTRACT: 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 kLa and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal kLa correlation.
A Respiration Activity Monitoring System (RAMOS) was used to measure kLa values in cylindrical disposable shaken bioreactors and Buckingham's pi-Theorem was applied to define a dimensionless equation for kLa. In this way, a scale- and volume- independent kLa 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.
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 kLa correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.
Journal of Biological Engineering 12/2013; 7(1):28.
[Show abstract][Hide abstract] ABSTRACT: 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 1500L 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 1500L and that the inner-to-shaking diameter is a suitable scale-up factor for mixing.
[Show abstract][Hide abstract] ABSTRACT: To achieve rapid and cost-effective expression of monoclonal antibodies for preclinical tests, we combined PEI mediated transient
gene expression with large-scale shake cultivation systems. Here we present tests performed at the 50-L scale that resulted
in the production of more than half a gram of IgG from CHO cells in a single transient batch process. This suggests that shake
bioreactor systems might be ideally suited for a variety of cell culture based processes aimed at efficiently expressing recombinant
[Show abstract][Hide abstract] ABSTRACT: In this paper a novel and easily applied method to measure the mass transfer resistance of the sterile closures (e.g. cotton plug) of shaken bioreactors is introduced. This method requires no investment in special equipment (e.g. an oxygen sensor) and can be performed with the materials usually available in typical laboratories. The method is based on the model of Henzler et al. (1986), which mechanistically describes mass transfer through the sterile closure of a shaken bioreactor based on diffusion coupled with Stefan convection. The concentration dependency of the multi-component diffusion coefficients is taken into account. The water loss from two equivalent shaken bioreactors equipped with sterile closures during several days of shaking is measured. One flask contains distilled water, the other a saturated salt solution. From the water evaporation rate in each of the two flasks, the new model presented calculates the relative humidity in the environment, the average diffusion coefficient of oxygen in the sterile closure (D(O2)), and the diffusion coefficient of carbon dioxide (D(CO2)) . The diffusion coefficient of carbon dioxide (D(CO2)) only depends on the density and material properties of the sterile closure and not on the gas concentrations and is, therefore, an ideal parameter for the characterization of the mass transfer resistance. This new method is validated experimentally by comparing the diffusion coefficient of oxygen (D(O2)) to a measurement by the classic dynamic method; and by comparing the calculated relative humidity in the environment to a humidity sensor measurement.
Biotechnology and Bioengineering 01/2008; 98(5):999-1007. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.
Biotechnology and Bioengineering 11/2006; 95(3):433-45. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxygen supply is a key parameter in aerobic fermentation processes like the industrial production of amino acids. Although the oxygen transfer rate (OTR; or the volumetric oxygen transfer coefficient k(L)a) is routinely analyzed by engineers during stirred tank fermentations, it is often not taken into account by biologists conducting screening experiments in shake flasks. To show the importance of knowing how to avoid oxygen transfer limitations during primary screenings, Corynebacterium glutamicum ATCC 13032 (wild-type strain) and DSM 12866 (lysine-producing strain) were cultivated in shake flasks with different culture liquid volumes and under different shaking conditions. With the Respiration Activity Monitoring System, the OTR was determined quasi-continuously. Optical density as well as concentrations of lysine and byproducts (lactate, acetate, succinate) were determined off-line and correlated with the OTR signal. From the results, design criteria for improved screening in shaken bioreactors that help to avoid selection of suboptimal strains during early process development steps can be derived. Finally, the suitability of DSM 12866 as a strain for industrial processes with a high space-time yield is discussed.
Applied Microbiology and Biotechnology 11/2006; 72(6):1157-60. · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biotechnology and Bioengineering 04/2005; 89(6):698-708. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Online measurement of respiration activity (including oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), respiratory quotient (RQ)) of microbial cultures in stirred bioreactors with exhaust gas analysis has been state of the art for years. As much more experiments are conducted in shaking bioreactors compared to stirred bioreactors, Anderlei and Buchs [Biochem. Eng. J. 7 (2001) 157] developed a measuring device (OTR-Device) for online determination of the oxygen transfer rate in shake flasks under sterile conditions. In this paper, an extension of the OTR-Device, termed respiration activity monitoring system (RAMOS) is described, which allows additional measurement of the carbon dioxide transfer rate and the respiratory quotient in shaking bioreactors. Fermentations of the yeasts Saccharomyces cerevisiae and Pichia stipitis carried out with RAMOS are presented. These measurements show very clearly the differences in respiration activities between the Crabtree-positive yeast S. cerevisiae and the Crabtree-negative yeast P stipitis. Furthermore, a fermentation of the bacterium Corynebacterium glutamicum is presented, showing the influence of an oxygen limitation on the metabolic activities of the culture. Also, a fermentation of a hybridoma cell line was carried out with RAMOS to elucidate the measuring sensitivity of the system. The new device provides the most important and characteristic parameters (OTR, CTR, RQ) representing biological cultures online, enabling users to draw conclusions on metabolisms of microorganisms already in shaking bioreactors. (C) 2003 Elsevier B.V. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Screening cultures are usually non-monitored and non-controlled due to a lack of appropriate measuring techniques. A new device for online measurement of oxygen transfer rate (OTR) in shaking-flask cultures was used for monitoring the screening of Hansenula polymorpha. A shaking frequency of 300 rpm and a filling volume of 20 ml in 250-ml flasks ensured a sufficient oxygen transfer capacity of 0.032 mol (l h)(-1) and thus a respiration not limited by oxygen. Medium buffered with 0.01 mol phosphate l(-1) (pH 6.0) resulted in pH-inhibited respiration, whereas buffering with 0.12 mol phosphate l(-1) (pH 4.1) resulted in respiration that was not inhibited by pH. The ammonium demand was balanced by establishing fixed relations between oxygen, ammonium, and glycerol consumption with 0.245+/-0.015 mol ammonium per mol glycerol. Plate precultures with complex glucose medium reduced the specific growth rate coefficient to 0.18 h(-1) in subsequent cultures with minimal glycerol medium. The specific growth rate coefficient increased to 0.26 h(-1) when exponentially growing precultures with minimal glycerol medium were used for inoculation. Changes in biomass, glycerol, ammonium, and pH over time were simulated on the basis of oxygen consumption.
Journal of Industrial Microbiology and Biotechnology 11/2003; 30(10):613-22. · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel.
[Show abstract][Hide abstract] ABSTRACT: One of the mass transfer resistances for the gas exchange of shaking flasks is the sterile plug. The gas exchange through the sterile plug is described by an extended model of Henzler and Schedel [Bioprocess Eng. 7 (1991) 123]. Based on this model, a new method was developed to obtain the mass transfer resistance of various sterile closures. It consists of measuring the water evaporation rate of the shaking flask and is therefore very easily applied. Sterile plugs made of cotton, wrapped paper, urethane foam and fibreglass and caps made out of aluminium and silicone have been examined. Instead of the oxygen transfer coefficient (kO2), which is commonly found in the literature, the carbon dioxide diffusion coefficient (DCO2) is used to describe the mass transfer resistance of the sterile plug. The investigation revealed that this resistance is mainly dependent on the neck geometry and to a lesser extent on the plug material and density. The gas exchange of aluminium-caps was not reproducible.