Methods for identifying precipitates and improving stability of chemically defined highly concentrated cell culture media

Abstract Currently, within the biopharmaceutical industry, media development is a key area of development as the ratios and concentrations of media components such as amino acids, metals, vitamins, sugars, salts, and buffering agents play arguably the largest role in cellular productivity and product quality. However, optimizing media for these targets often conflicts with solubility limitations and slow‐rate chemical reactions that result in precipitation formation. Here we present methods such as inductively coupled plasma mass spectrometry (ICP‐MS), X‐ray fluorescence (XRF), colorimetry, and turbidity to identify multiple likely components of a complex precipitate that was observed in preparations of a custom nutrient feed medium across all storage conditions evaluated. Using these analytical methods, as well as adjustments to the formulation pH, increasing the pyruvate concentration, and removing sodium bicarbonate, we were able to extend the media shelf life from approximately 10 days to over 28 days. Alternatively, copper, selenium, and magnesium sources were removed from the media and no precipitation was observed until 32 days after prep, pointing to key metals as the probable root cause of precipitation. By analytically quantifying the precipitate using the methods above, instead of visual inspection, which is the current industry standard for media precipitation observation, we were better able to compare conditions to one another and relate them to the onset of precipitation. Cell culture performance and product quality remained comparable to the historical process despite the media formulation changes.