Non‐invasive and time‐resolved measurement of the respiration activity of Chinese hamster ovary cells enables prediction of key culture parameters in shake flasks

Abstract

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.

Introducing oxygen transfer rate measurements as a novel method for time-resolved cytotoxicity assessment in shake flasks

“…However, as the primary focus of the study was not to provide improved culture media for cytotoxicity assessment, no change to the culture medium was made. This way a comparison to previously published studies [28,29] is still possible. Directly before use, the medium was supplemented with 6 mM l-glutamine (Gibco Life Sciences, Thermo Fisher Scientific, USA) and 1% (v/v) PenStrep (stock with 10,000 Units mL −1 penicillin and 10 g L −1 streptomycin) (Sigma-Aldrich, USA).…”

“…The oxygen uptake rate (OUR) provides a wide variety of information during mammalian cell culture cultivation [23,28,29,41]. As oxygen consumption is directly linked to cell metabolism, the OUR provides information on culture viability.…”

“…In slow mammalian cell cultures this is always the case, and the OUR may be determined from measurement of the OTR. Measurement of the OTR of CHO suspension cells has been realized in shake flasks using a prototype device of the Respiration Activity Monitoring System (RAMOS) [28,29] as well as a commercially available Transfer-Rate Online Measurement (TOM) device [29]. Both systems utilize electrochemical sensors to determine the OTR.…”

“…However, parallelization is only limited by the number of flasks that can be operated in the incubator simultaneously. Even at the comparably low breathing activity of CHO cells, up to one OTR value per hour can be obtained [28,29]. For CHO suspension cells, information on the VCC and differences in culture behavior at different initial cell concentrations were determined using RAMOS [29].…”

“…Cryopreservation was performed as described in detail by Ihling et al [28] (cell line one) and (cell line two) [29] and according to the basic principles described, for example, by Freshney [20]. After thawing, one washing step was included to remove DMSO.…”