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.
To yield high concentrations of protein expressed by genetically modified Escherichia coli, it is important that the bacterial strains are cultivated to high cell density in industrial bioprocesses. Since the expressed target protein is mostly accumulated inside the E. coli cells, the cellular product formation can be directly correlated to the bacterial biomass concentration. The typical way to determine this concentration is to sample offline. Such manual sampling, however, wastes time and is not efficient for acquiring direct feedback to control a fedbatch fermentation. An E. coli K12-derived strain was cultivated to high cell density in a pressurized stirred bioreactor on a pilot scale, by detecting biomass concentration online using a capacitance probe. This E. coli strain was grown in pure minimal medium using two carbon sources (glucose and glycerol). By applying exponential feeding profiles corresponding to a constant specific growth rate, the E. coli culture grew under carbon-limited conditions to minimize overflow metabolites. A high linearity was found between capacitance and biomass concentration, whereby up to 85 g/L dry cell weight was measured. To validate the viability of the culture, the oxygen transfer rate (OTR) was determined online, yielding maximum values of 0.69 mol/l/h and 0.98 mol/l/h by using glucose and glycerol as carbon sources, respectively. Consequently, online monitoring of biomass using a capacitance probe provides direct and fast information about the viable E. coli biomass generated under aerobic fermentation conditions at elevated headspace pressures.
Escherichia coli producing a plasmid DNA (pDNA) vaccine was cultivated in fed‐batch mode at small scale (1 L) using oxygen‐enriched air, and at pilot scale (50 L) using a pressurized bioreactor, to maintain aerobic conditions. In the small scale, the attained oxygen transfer rate (OTRMAX) using an oxygen concentration in the inlet gas of 68.2%, reached 0.42 mol L−1 h−1. The OTRMAX in the pressurized reactor with an overpressure of 8 bar was 0.5 mol L−1 h−1. In the small‐ and pilot‐scale cultivations, the final biomass concentrations (74 and 79 g/L, respectively), pDNA concentrations (236 and 215 mg/L), overall productivity and pDNA topology were very similar. Therefore, the pressurized cultivation is a viable option to scale up pDNA production processes.
A systematic and integrated use of single‐use technologies was combined with a robust monoclonal antibody platform, which led to a substantial reduction of manufacturing costs, reduced timelines and increased flexibility in clinical manufacturing. A direct scale‐up of a high titer monoclonal antibody‐expressing CHO DG44‐based cell culture platform was performed from shake flasks to a 1000 L production scale in a completely single‐use manufacturing facility. The scale‐up was done on the basis of calculating the specific volumetric power input which allowed a direct transfer from small culture volumes to the production scale. The timelines for process development were reduced to 3 months from the Research Cell Bank to the drug substance with highly optimized cells and appropriate culture conditions.
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