Characterisation of Shake Flasks for Cultivation of Animal Cell Cultures

Boehringer Ingelheim Veterinary Research Center Gmbh & Co. Kg, Hanover, Lower Saxony, Germany
DOI: 10.1007/978-1-4020-5476-1_131

ABSTRACT This study investigated the oxygen transfer processes and general correlations between culture performance and the operating
conditions for shake flask fermentations of animal cell cultures. This involved both the online measurement of the oxygen
transfer rate and the continuous recording of measurements for the dissolved oxygen concentration in the shake flask.

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    ABSTRACT: BACKGROUND: The Respiration Activity Monitoring System (RAMOS) is an established device to measure on-line the oxygen transfer rate (OTR), thereby, yielding relevant information about metabolic activities of microorganisms and cells during shake flask fermentations. For very fast-growing microbes, however, the RAMOS technique provides too few data points for the OTR. Thus, this current study presents a new model based evaluation method for generating much more data points to enhance the information content and the precision of OTR measurements. RESULTS: In cultivations with E.coli BL21 pRSET eYFP-IL6, short diauxic and even triauxic metabolic activities were detected with much more detail compared to the conventional evaluation method. The decline of the OTR during the stop phases during oxygen limitations, which occur when the inlet and outlet valves of the RAMOS flask were closed for calibrating the oxygen sensor, were also detected. These declines reflected a reduced oxygen transfer due to the stop phases. In contrast to the conventional calculation method the new method was almost independent from the number of stop phases chosen in the experiments. CONCLUSIONS: This new model based evaluation method unveils new peaks of metabolic activity which otherwise would not have been resolved by the conventional RAMOS evaluation method. The new method yields substantially more OTR data points, thereby, enhancing the information content and the precision of the OTR measurements. Furthermore, oxygen limitations can be detected by a decrease of the OTR during the stop phases.
    Journal of Biological Engineering 08/2012; 6(1):11. DOI:10.1186/1754-1611-6-11
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    ABSTRACT: Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.
    Biotechnology Progress 01/2012; 28(1):188-95. DOI:10.1002/btpr.729 · 1.88 Impact Factor
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    ABSTRACT: Selecting the right design of bioreactors is crucial for guaranteeing the reproducibility of bioprocesses. Up to now, conventionally designed bioreactors consist typically of melted or clamped joints. Since melting of borosilicate glass leads to large deformed areas along the joint, the desired geometric reproducibility is not ensured. Moreover, clamping complicates and greatly restricts the bioreactor design. Bonding, how-ever, is advantageous in that it does not alter the ma-terial joined and it is easy to use. Furthermore, it has been recently shown that specially developed glass bonding techniques withstand multiple autoclaving cycles. The current research investigated practice-relevant parameters influencing the lifespan of ep-oxy-or (urethane) acrylate-bonded glass bioreactors. Hereby, the influence of cleaning and sensitivity to fermentation compounds (ethanol and acetic acid) was quantified using glass-glass and glass-stainless steel specimens. Whereas cleaning did not adversely affect the durability of glass bonds, high concentra-tions of the fermentation compounds ethanol and acetic acid resulted in accelerated corrosion and sub-sequent bond failure. Moreover, no effect of eight different epoxy and (urethane) acrylate adhesives was observed on selected model organisms Escherichia coli K12 and Hansenula polymorpha wild type. Another objective of this study was to refine the design of two small-scale bioreactors (ca. 250 mL) by replacing clamps and melted joints by adhesive joints. It was found that the bonded bioreactors yielded a higher geometric reproducibility than that of conventional melted or clamped ones. In conclusion, bonded glass joints greatly enhance the geometric reproducibility of bioreactors and, in turn, the reproducibility of bioprocesses. As glass bonding is easy to handle, it opens up new opportunities to design bioreactors that had been previously too expensive and complicated.
    Advances in Bioscience and Biotechnology 01/2011; 2:233-243.