Investigating trace metal precipitation in highly concentrated cell culture media with Pourbaix diagrams

Abstract

Commercial production of therapeutic proteins using mammalian cells requires complex process solutions, and consistency of these process solutions is critical to maintaining product titer and quality between batches. Inconsistencies between process solutions prepared at bench and commercial scale may be due to differences in mixing time, temperature, and pH which can lead to precipitation and subsequent removal via filtration of critical solution components such as trace metals. Pourbaix diagrams provide a useful tool to model the solubility of trace metals and were applied to troubleshoot the scale‐up of nutrient feed preparation after inconsistencies in product titer were observed between bench‐ and manufacturing‐scale batches. Pourbaix diagrams modeled the solubility of key metals in solution at various stages of the nutrient feed preparation and identified copper precipitation as the likely root cause of inconsistent medium stability at commercial scale. Copper precipitation increased proportionally with temperature in bench‐scale preparations of nutrient feed and temperature was identified as the root cause of copper precipitation at the commercial scale. Additionally, cell culture copper titration studies performed in bench‐scale bioreactors linked copper‐deficient mammalian cell culture to inconsistent titers at the commercial scale. Pourbaix diagrams can predict when trace metals are at risk of precipitating and can be used to mitigate risk during the scale‐up of complex medium preparations.

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

“…that map the most stable metal species in an aqueous system, are readily used in evaluating corrosion and could be adopted in the biopharmaceutical industry to better predict solution stability. 31,32 For this work, Pourbaix diagrams were used to validate our empirical findings and confirmed copper and selenium as potentially precipitating species (data not shown). However, due to the complex nature of these nutrient feed formulations and cell culture formulations in general, more specific characterization work is needed to increase the accuracy of the models.…”

“…Further characterization of the precipitation reactions could include re‐filtering the media after precipitation has occurred and using those concentrations as the baseline for a new nutrient feed solution. Pourbaix diagrams, which are electrode‐pH phase diagrams that map the most stable metal species in an aqueous system, are readily used in evaluating corrosion and could be adopted in the biopharmaceutical industry to better predict solution stability 31,32 . For this work, Pourbaix diagrams were used to validate our empirical findings and confirmed copper and selenium as potentially precipitating species (data not shown).…”