Cultivations of mammalian cells are routinely conducted in shake flasks. In contrast to instrumented bioreactors, reliable options for non-invasive, time-resolved monitoring of the culture status in shake flasks are lacking. The Respiration Activity Monitoring Respiration Activity Monitoring System system was used to determine the oxygen transfer rate (OTR) in shake flasks. It was proven that the OTR could be regarded as equal to the oxygen uptake rate as the change of the dissolved oxygen concentration in the liquid phase over time was negligibly small. Thus, monitoring the oxygen transfer rate (OTR) was used to increase the information content from shake flask experiments. The OTR of a Chinese hamster ovary cell line was monitored by applying electrochemical sensors. Glass flasks stoppered with cotton plugs and polycarbonate flasks stoppered with vent-caps were compared in terms of mass transfer characteristics and culture behavior. Similar mass transfer resistances were determined for both sterile closures. The OTR was found to be well reproducible within one experiment (standard deviation <10%). It correlated with changes in cell viability and depletion of carbon sources, thus, giving more profound insights into the cultivation process. Culture behavior in glass and polycarbonate flasks was identical. Monitoring of the OTR was applied to a second culture medium. Media differed in the maximum OTR reached during cultivation and in the time when all carbon sources were depleted. By applying non-invasive, parallelized, time-resolved monitoring of the OTR, the information content and amount of data from shake flask experiments was significantly increased compared to manual sampling and offline analysis. The potential of the technology for early-stage process development was demonstrated.

Background: Shake flasks are frequently used for mammalian cell suspension cultures. For process development and routine culture monitoring, information on culture behavior is needed early on. Main methods and major results:Here, cell-specific oxygen uptake rates (qO 2 ) of two CHO cell lines were determined from shake flask experiments by simultaneous measurement of oxygen transfer rates (OTR) and viable cell concentrations (VCC). For cell line one, qO 2 decreased from 2.38⋅10 −10 to 1.02⋅10 -10 mmol cell −1 h −1 during batch growth. For cell line two, qO 2 was constant (1.90⋅10 −10 mmol h −1 ). Determined qO 2 values were used to calculate the VCC from OTR data. Cumulated oxygen consumption and glucose consumption were correlated for both cell lines and enabled calculation of glucose concentrations from OTR data. IgG producing cell line one had an oxygen demand of ∼15 mmol oxygen g glucose −1 , cell line two consumed ∼5 mmol oxygen g glucose −1 .The established correlations for determination of VCC and glucose were successfully transferred to subsequent cultivations for both cell lines. Combined measurement of the OTR and the carbon dioxide transfer rate enabled quantitative determination of the lactate concentration (production and consumption) without sampling. Conclusions and implications:Taken together, non-invasive measurement of the respiration activity enabled time-resolved determination of key culture parameters for increased process understanding in shake flasks. K E Y W O R D Scarbon dioxide transfer rate, Chinese hamster ovary cells, oxygen transfer rate, respiratory quotient, shake flasks INTRODUCTIONChinese hamster ovary (CHO) cells are the most important mammalian cells used for the stable production of therapeutic proteins. [1] Even though traditionally applied for microbial cultivations, shake flasks areThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.