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.

Background Determining the cytotoxicity of test substances is essential for the safety assessment of chemicals. To quantify the cytotoxicity, dose–response curves are determined and the half-maximum inhibitory concentration (IC50) is subsequently calculated. Results often rely on a single endpoint evaluation which typically requires manual sampling and subsequent sample analysis to determine the IC50. Hence, no information on culture behavior are available during treatment. Here, measurement of the oxygen transfer rate (OTR) was applied as a method for time-resolved assessment of the cytotoxicity to (a) provide information on culture behavior during treatment and (b) quantitatively assess the cytotoxic effect of a test substance. Results To investigate the applicability of the method, different types and concentrations of test substances were added and the OTR was measured for two different CHO suspension cell lines in shake flasks: (a) For CHO cell line one, culture behavior was not affected when 0.1 mM ZnCl2 was added. However, for both cell lines, adding up to 0.2 mM CoCl2 led to a decrease in the OTR that was concentration dependent. For CHO cell line two, adding up to 5% DMSO led to a concentration-dependent decrease in the OTR and in the slope of the OTR; (b) From the measured OTR, dose–response curves were established and used to calculate the IC50. For cell line one, the IC50 for CoCl2 was 0.03 mM while the IC50 from a comparative measurement based on trypan blue exclusion was 0.06 mM. Cell line two was less susceptible to CoCl2 as the IC50 was not reached at the concentrations tested. For DMSO, single OTR values and determined slopes were used for evaluation. In both cases, the IC50 was calculated to 2.3%. Conclusions Our approach provides a method for analyzing the cytotoxicity of a test substance based on OTR measurements. This method provides (i) insights on culture behavior, (ii) information on the progression of cytotoxicity, (iii) dose–response curves, and (iv) a first indication of IC50 values. Quantitative assessment of cytotoxicity is possible non-invasively and in real-time during treatment. Compared to traditional endpoint evaluation, our method simplifies experimentations and enhances the comparison of assay results.

Lentiviral vectors (LVs) are widely used in clinical trials of gene and cell therapy. Low LV stability incentivizes constant development and the improvement of gentle process steps. Steric exclusion chromatography (SXC) has gained interest in the field of virus purification but scaling up has not yet been addressed. In this study, the scaling up of lentiviral vector purification by SXC with membrane modules was approached. Visualization of the LVs captured on the membrane during SXC showed predominant usage of the upper membrane layer. Furthermore, testing of different housing geometries showed a strong influence on the uniform usage of the membrane. The main use of the first membrane layer places a completely new requirement on the scaling of the process and the membrane modules. When transferring the SXC process to smaller or larger membrane modules, it became apparent that scaling of the flow rate is a critical factor that must be related to the membrane area of the first layer. Performing SXC at different scales demonstrated that a certain critical minimum surface area-dependent flow rate is necessary to achieve reproducible LV recoveries. With the presented scaling approach, we were able to purify 980 mL LVs with a recovery of 68%.