The carbon dioxide/bicarbonate system is routinely used as a buffer in mammalian cell culture medium. However, due to the continuous degassing of carbon dioxide and its dependence on temperature, it is difficult to achieve the target pH during preparation at ambient temperature and without control of dissolved carbon dioxide, and even more so at cell culture operating conditions. These problems were amplified when preparing multiple (e.g., 20) customized preparations of an in‐sourced proprietary medium during research and process development of mammalian cell culture systems. Thus, a mathematical model was created to specify the amount of acid or base to add during preparation so as to achieve the target pH of each medium at process conditions without having to do titration. The relationship between gaseous carbon dioxide and the dissolved carbon dioxide in the proprietary medium containing unknown species was specified using a modified Henry's law equation (as done in prior work). Further, to allow medium preparation without doing titration, the acid/base properties of the proprietary medium were fitted by a parameter related to its Net Medium Acids during specification of model parameters. A subset of the solutions was prepared and tested in bioreactors with controlled CO2 flow to validate the model. Then, the model was used to assess the equivalence of the pHs of the customized medium formulations despite variations in pCO2 that occurred during incubation and sampling.

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