Time-Resolved Monitoring of the Oxygen Transfer Rate of Chinese Hamster Ovary Cells Provides Insights Into Culture Behavior in Shake Flasks

Lara Pauline Munkler,Britta Reichenbächer,Nina Ihling,Johannes Wirth,Jochen Büchs,Roland Wagner,Dietmar Lang,Christoph Berg

doi:10.3389/fbioe.2021.725498

Lara Pauline Munkler, Britta Reichenbächer + Show 6 more

Open Access

https://doi.org/10.3389/fbioe.2021.725498

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Abstract

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.

Highlights

Complex therapeutic proteins are dominantly produced using mammalian cell cultivation with Chinese hamster ovary (CHO) cells as the preferred host (Wurm, 2004)
For 250-ml Erlenmeyer flasks stoppered with cotton plugs, the sterile closure’s mass transfer resistance was much smaller than the gas-liquid mass transfer resistance (Mrotzek et al, 2001; Anderlei et al, 2007)
For the vent-cap of the singleuse polycarbonate flask, kst,O2 was determined to 1.17·10−5 mol s−1. This value aligns well with the wide- and narrow-necked flasks stoppered with cotton plugs described by Mrotzek et al (2001)

Summary

Introduction

Complex therapeutic proteins are dominantly produced using mammalian cell cultivation with Chinese hamster ovary (CHO) cells as the preferred host (Wurm, 2004). While manufacturing is usually carried out in large bioreactors, early-stage process development relies on smaller devices of different scales. Manufacturers can choose from a broader range of different small-scale cultivation systems for mammalian cell cultivations the information content from these systems is less. Time-Resolved Monitoring of CHO Cells compared to highly instrumentalized bioreactors (Betts and Baganz, 2006; Bareither and Pollard, 2011). Microtiter plates can be used for cultivation at an early stage of process development (Girard et al, 2001; Deshpande and Heinzle, 2004). Substantial evaporation is a significant drawback of very small cultivations systems, during long-term cultivations necessary for bioprocess development studies (Betts and Baganz, 2006).

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