Optimizing Bispecific Antibody Expression via Multi‐Omics Analysis and Vector Redesign

Bispecific antibodies are a growing class of therapeutics that simultaneously engage two targets. However, their complex molecular structures pose challenges for production in Chinese hamster ovary cells, the current industry standard for biologics manufacturing. Here we present a case study of three IgG-scFv format bsAbs expressed in CHO cells, in which one candidate exhibited markedly lower titers despite high sequence homology to the other two. Using multi-omics analysis (RNA sequencing, splicing prediction, codon optimization assessment, and motif screening) to investigate potential causes, we identified several likely mechanisms for poor expression, including aberrant splicing motifs, ribosome pausing sites, and suboptimal codon usage. Through targeted protein and DNA sequence engineering, we generated a revised variant with an 11-fold increase in stable expression titers. This work demonstrates that integrating sequence-level bioinformatic and synthetic biology diagnostics can directly improve manufacturability, providing a generalizable framework for resolving hidden expression liabilities in complex biologics.