Rational design of dosage compensated expression vectors for multi‐subunit biologics manufacturing

Abstract Chinese hamster ovary (CHO) cells are the predominant platform for biologics production. Expression of multi‐subunit proteins within a single CHO cell requires balanced subunit expression to achieve optimal product quality and yield. Through systematic analysis of CHO expression data generated at the discovery stage using co‐transfected monocistronic vectors, we found that subunit expression balance strongly correlates with product purity across both monoclonal and bispecific antibodies. These insights enabled the rational design of multicistronic vectors for production cell line development. For bispecific antibodies containing Fab and single‐chain variable fragment (scFv) arms, scFv chains were frequently overexpressed, contributing to reduced product purity. Increasing heavy chain (HC) expression, for example through the introduction of an additional HC gene copy, improved both purity and titer across multiple programs. Likewise, optimizing the relative positioning of knob and hole HC genes within bicistronic vectors helped balance chain expression and enhance product purity in a complex IgG‐like molecule. Overall, early‐stage monocistronic expression analysis provides a framework for rational vector design of multi‐subunit biologics, enabling more efficient cell line development with the potential to accelerate preclinical timelines and reduce resource utilization.