Boehringer Ingelheim Veterinary Research Center Gmbh & Co. Kg, Hanover, Lower Saxony, Germany
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.
"These cultivations were carried out in a Respiration Activity MOnitoring System (RAMOS)   to monitor possible adverse effects of the adhesive on the metabolic activity of the microbes. RAMOS has already been successfully used for several investigations       . Adhesive disks (see Table 1) were produced as disks having a diameter of 15 mm and a height of 1 mm. "
[Show abstract][Hide abstract] 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(04):233-243. DOI:10.4236/abb.2011.24034
[Show abstract][Hide abstract] ABSTRACT: Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake-flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake-flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS-CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake-flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered.
[Show abstract][Hide abstract] ABSTRACT: Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines.
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