P40: Identification and prevention of a colored product variant during recombinant protein production in E. coli

During late stage clinical development of an Escherichia coli (E. coli) recombinant protein production process, changes to the fermentation process were implemented to increase productivity, eliminate animal-derived media components and improve process consistency.  Significant changes were also implemented for the purification process to improve plant fit, remove a prominent product variant and increase throughput.  The optimized production process was developed using a small scale model via univariate and multivariate studies and was successfully transferred to manufacturing scale.  During manufacturing scale qualification, two Drug Substance lots appeared slightly brown in color and did not meet the expected Yellow series color criterion for the Clarity, Opalescence, and Coloration (COC) assay. Extensive investigational studies determined that the coloration was caused by an adduct formed at trace levels via cysteine residues on the recombinant product. The compound modifying the recombinant product was identified as 1,4-dihydroxy-2-naphthoate (DHNA), a metabolite derived from E. coli cells.  The investigation to identify the colored species, the root cause, and the development of a control strategy to prevent the formation of the brown colored product variant, will be discussed.