Drug substance (DS) color is an important quality attribute for release, stability and comparability studies of therapeutic proteins. With the increase of DS concentrations and biologics pipelines made in chemically defined media, atypical DS color other than colorless or pale yellow has been recently reported in the biopharmaceutical industry. This presentation reports an investigative approach to elucidate the root cause of a brown colored drug substance observed in manufacturing using CHO cells. Although analytical characterization data indicated that the brown color DS had no major quality issue, it is necessary to find the root cause and reduce DS color to ease placebo design for clinical use. It was demonstrated that the brown color was caused by the chemically defined medium containing high levels of iron, vitamin B12 (VB12), and copper regardless of cell lines. Iron caused tryptophan oxidation in the protein to form N-formylkynurenine and kynurenine products, which likely contributed to a yellow DS color. A pink DS color was caused by the residual VB12 bound to DS. The brown color was the result of the combinatory effect of yellow and pink colors. In addition, high levels of iron in the basal medium promoted cell growth and protein titer, while high levels of VB12 had no impact on cell culture performance. Copper neither caused any DS color nor improved titer. Finally a modified basal medium was developed to produce a pale yellow DS in manufacturing. To our knowledge, this study advances knowledge of the mechanism of DS color formation and cell culture manufacturing processes for therapeutic proteins.

Iron mediated tryptophan oxidation and vitamin B12 carryover caused a brown color formation of protein drug substance manufactured in CHO cells using chemically defined media

247th National Meeting (2014)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.