Systematic Evaluation of Vector Sequence Elements in Isogenic CHO Cells for Improved Antibody Production

Monoclonal antibodies (mAbs) constitute a leading class of biotherapeutics, and meeting global demand requires manufacturing platforms that deliver rapid and reliable expression. Nevertheless, efficient and predictable production remains challenging because multiple vector elements can substantially influence productivity and product quality. In this study, we examined how sequence design features influence expression in a targeted-integration CHO system using six antibodies (A-F). We evaluated codon usage in the constant light (CL) and constant heavy (CH) regions with and without LALA/YTE Fc mutations, the type of signal peptide, codon optimization of the variable regions, and substitution of the kappa light chain with lambda. We found that changing codons of CL and CH to suboptimal codons reduced expression for all six antibodies, independent of LALA/YTE status. Replacing chain-specific signal peptides with a single identical peptide produced antibody-specific outcomes: productivity decreased for four antibodies (A, C, D, and E), increased for antibody B by ∼1.5-fold, and remained unchanged for antibody F. In addition, codon optimization of the variable regions generally enhanced productivity in an antibody-dependent manner, with improvements ranging from ∼2-fold to ∼18-fold. Finally, substituting kappa with lambda decreased productivity for three antibodies, suggesting a CHO cell preference for the kappa isotype. Collectively, these findings delineate practical sequence-engineering principles for CHO expression, prioritize codon usage in constant and variable domains, maintain chain-appropriate signal peptides, and account for kappa/lambda dependencies, thereby improving construct selection and accelerating development of high-yielding mAb producers.