Impact of Heavy Chain C‐Terminal Codons on Recombinant Monoclonal Antibody Expression Levels

ABSTRACT Advances in process optimization and stable CHO cell line engineering have enabled recombinant monoclonal antibody (mAb) titers above 10 g/L in fed‐batch cultures. Despite these advances, the correlation between mAb nucleotide coding sequence and protein yield is still not fully understood. Initial studies with two IgG1 mAbs showed that Lys codon selection at the heavy chain (HC) C‐terminus significantly affected yields in certain sequence contexts, as the titer of mAb1 was unchanged by codon selection, while mAb2 titer dropped four‐fold with AAG Lys compared to AAA Lys . To determine the factors contributing to this difference, CHO cell lines expressing the two mAb variants with different HC C‐terminal variants were generated. The specific C‐terminal codon pair GGT Gly –AAG Lys notably reduced HC mRNA transcript levels and mAb yields in CHO expression studies. Metabolic labeling studies using a modified uracil analog revealed that the decreased HC transcript levels resulted from reduced accumulation of nascent HC mRNA. Furthermore, the C‐terminal codon pair GGT Gly –AAG Lys was found to broadly influence the expression of other industrially relevant IgG mAb isotypes, including IgG2 and IgG4. This study establishes a connection between the HC C‐terminal nucleotide sequence and mAb expression titer, offering insights for optimizing the expression of Fc‐containing molecules.