Improved mass transfer allows selection of high performers in early stage development

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1 Improved mass transfer allows selection of high performers in early stage development Small scale systems for suspension cultures, such as standard orbital shakers are widely used in cultivating micro-organisms. Especially during the selection of new potential highproducers an orbital shaker cultivation is used to find the possible candidates. Despite the widespread use, the oxygen limitation is often a major problem cultivation / fermentation. The organism selected based on Shaker cultivation might not be the best producer under the non (or less) oxygen limited conditions in the fermenter. Typically a different medium is used in the shakes flask cultivation compared to the fermenter medium. The shake flask medium contains less nutrients based on the assumption that the organism will not use all nutrient when cultivated in an orbital shaker. The Fermenter medium is a richer medium which can result in more biomass and higher product concentrations. We are convinced that using the fermenter medium in the shake flask level in combination with better mass transfer will give better predictions of what a specific organism can do on production scale in a fermenter. The aim of this study is the description of growth characteristics of different microbial systems. Therefore a standard shaking incubator is compared to the new Applikon fermentation system RAMbio. ResonantAcoustic Mixing ResonantAcoustic Mixing is a new approach to mixing and dispersion of materials ranging from nanoparticle suspensions to viscous gels. It is distinct from conventional impeller agitation found in a planetary mixer or speed mixer as well as ultrasonic mixing. Low frequency, high-intensity acoustic energy is used to create a uniform shear field throughout the entire mixing vessel. The result is rapid fluidization (like a fluidized bed) and dispersion of material. The mixing benefits are applicable to a wide variety of compounds. We have integrated the acoustic mixing into an incubator with temperature and humidity control. The mixing power is measured and controlled in the amount of G-force applied to the culture. RAMbio acoustic mixer incubator How ResonantAcoustic Mixing Works Page 1

2 ResonantAcoustic Mixing introduces acoustic energy into liquids, slurries, powders and pastes. An oscillating mechanical driver creates motion in a mechanical system comprised of engineered plates, eccentric weights and springs. This energy is then acoustically transferred to the material to be mixed. The underlying technology principle is that the system operates at resonance. In this mode, there is a nearly complete exchange of energy between the mass elements and the spring elements in the mechanical system. In a ResonantAcoustic mixer, the only element that absorbs energy (apart from some negligible friction losses) is the mix load itself. Thus, the ResonantAcoustic Mixer provides a highly efficient way of transferring mechanical energy directly into the mixing materials. As an example, a ResonantAcoustic mixer using 40 HP would require over 770 HP in a nonresonant equipment configuration to mix the same load. The vertical mixing movement of the RAMbio generates a pumping motion in the silicone stopper that force gas in and out of the shake flask through the gasfilter on top of the stopper. In contrast to other vented flask closures, Oxy-Pump Stoppers actively pump fresh air into shake flask cultures. When used in the RAMbio, Oxy-Pump Stoppers deliver up to a 2VVM headspace refreshment rate to flasks a rate equivalent to stirred-tank bioreactor platforms. Page 2

3 Experimental results To determine the influence of the ResonantAcoustic Mixing (RAM) technology on the performance of microbial cultures we have tested different cultures in parallel in an orbital shaker and in the RAMbio. To analyze the system performance and influence on different microbial strains, the yeast Pichia pastoris pink and the bacterium Escherichia coli BL21 were cultivated each in the two different complex media YPD and LB with glucose as major carbon source starting from the same pre-culture. For each culture 200 ml medium was used in a 500 ml shaking flask. The orbital shaker was set to 300 rpm being equivalent to 15 x g set for the RAMbio. Every hour a sample has been taken and the optical density (OD) at 600 nm wavelength and the glucose concentration has been determined. Page 3

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5 Conclusion The results do not show significant differences between the cultivation parameters in an orbital shaker or a resonance acoustic mixer for E.coli during the first six hours, for P.pastoris during the first eight hours of cultivation. After this first period the growth and the glucose consumption of the cultures on the orbital shaker come to rest whilst the growth and glucose consumption of the cultures with YPD-medium on the RAMbio still increase. The reason is most likely a significant improved oxygen supply with the resonance acoustic mixing technology, provided that there is a sufficient amount of carbon source available like in the YPD-medium. This proves that using a richer fermenter medium in the shake flask stage of process development will give more representative results of what can be expected in a later stage of process development or production. Applications of the technology are reducing cultivation time due to higher productivity in a shorter time and selection of the highest producer in early stage development. Data is generated by the group of Prof. P. Czermak at the Institute of Bioprocess Engineering and Pharmaceutical Technology of the University of Applied Sciences, Mittelhessen, Germany. Page 5