Identification of copper as a cell culture media component causing metabolite depletion and product sequence variants

The level of peptide sequence variants in a biologic drug substance batch is a critical product quality attribute that should be monitored and controlled. These sequence variants are typically caused by DNA single nucleotide variants that arise in cloning and amplification, mistranscription due to unstable vector DNA or cell age/production stresses, or mistranslation via tRNA wobble or mischarging. In this work, a low frequency of monoclonal antibody sequence variants was detected by mass spectrometry in a drug substance batch. The variants were distributed throughout the heavy and light chains at average levels of under 1% per site with no apparent codon bias. No product-coding DNA mutations were detected via deep sequencing data. This pattern of low level, widely-distributed variation strongly suggested a misincorporation mechanism via mischarging of aminoacyl-tRNA, presumably due to amino acid depletion during the process. Copper is a critical cell culture media component that can be modulated in fed-batch processes to induce lactate consumption via its role as a cofactor for mitochondrial function and respiration. However, complete consumption of lactate can also trigger reduced levels of other metabolites required for recombinant protein assembly, which can lead to product sequence variants. To investigate the potential relationship between media copper supplementation and sequence variants, various levels of copper were supplemented into the basal media for fed-batch cultures at the 250 mL bioreactor scale. Mass spectrometry analysis of the partially purified antibody indicated a positive correlation between the amount of copper supplemented and the level of detected sequence variants as well as a mechanism for sequence variant reduction via targeted nutrient feeding. This work has identified a potential mechanism of sequence variant generation related to cell culture media copper levels as well as process alterations to prevent such variation in future batches, highlighting the importance of carefully controlling trace metal levels. Additional studies may be required to validate the potential mechanism