Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells

Krattenmacher F (2020)
Bielefeld: Universität Bielefeld.

Bielefelder E-Dissertation | Englisch
 
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Autor*in
Krattenmacher, Florian
Betreuer*in
Noll, ThomasUniBi ; Bücheler, Uwe
Abstract / Bemerkung
Chemically defined media (CDM) for cell culture are routinely used in industrial processes for recombinant protein production from mammalian expression systems as for example Chinese hamster ovary (CHO) cells. As CDM are nowadays considered as the industry standard the focus has shifted from implementation and improvement of performance to additionally their chemical behavior and the impact on process robustness. Since CDM are highly concentrated aqueous mixtures of versatile chemical compounds one particular problem in this context is the high risk for chemical reactions and instability.
Therefore, a major focus of this thesis is the generation of understanding for chemical interactions of CDM compounds and especially the establishment of analytical technologies for the purpose of media characterization. Thus, a mixed mode liquid chromatography tandem mass spectrometry (LC-QqQ-MS) method that is able to simultaneously quantify the majority of media compounds has been developed and validated. This powerful method has been applied to characterize the chemical behavior of feed media under process relevant conditions as preparation and storage. Further on line and off line analytics have been applied to gain insight into CDM chemistry.
The application of probes measuring standard parameters have shown the dynamic behavior of chemical key parameters during CDM powder hydration. A Particle probe, such as the focused beam reflectance measurement (FBRM), has been shown to be useful for dissolution behavior investigations of different media recipes or powder compositions. However, it is rather difficult to establish the technology for batch to batch comparison or the monitoring of deviations from the standard preparation conditions. Media preparations with simplified media powders revealed that the compounds ascorbic acid and phosphates cause an apparent drop in dissolved oxygen concentration upon iron compound addition. The combination of the experiments with the newly developed LC QqQ MS method confirmed the comparability of chemical behavior in different media matrixes of most of the CDM compounds but highlighted some differences. Furthermore, measurements with the LC-QqQ-MS showed that the effect of preparation temperature and relevant storage conditions on media stability were negligible. In contrast, measurement of samples over storage time identified unstable compounds. A closer look at the media after storage showed that some formulations formed precipitate during storage and the collection of the solid material on filter membranes revealed their different appearance. Investigations of the material with specialized analytics proved that their identity was heterogeneous. One precipitate that was drawing attention on itself was of silver color and could be shown to consist of Sulphur.
Jahr
2020
Page URI
https://pub.uni-bielefeld.de/record/2943695

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Krattenmacher F. Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells. Bielefeld: Universität Bielefeld; 2020.
Krattenmacher, F. (2020). Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells. Bielefeld: Universität Bielefeld. doi:10.4119/unibi/2943695
Krattenmacher, F. (2020). Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells. Bielefeld: Universität Bielefeld.
Krattenmacher, F., 2020. Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells, Bielefeld: Universität Bielefeld.
F. Krattenmacher, Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells, Bielefeld: Universität Bielefeld, 2020.
Krattenmacher, F.: Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells. Universität Bielefeld, Bielefeld (2020).
Krattenmacher, Florian. Beyond chemically defined – Characterization of chemically defined cell culture medium for the cultivation of CHO cells. Bielefeld: Universität Bielefeld, 2020.
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