Abstract
Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity. Moreover, mispairing carries significant challenges for downstream purification, decreasing yields and increasing the cost of bioprocess development. In this study, quantitative transcriptomics and proteomics analyses were employed to investigate which signaling pathways correlated with low and high mispairing clone signatures. Gene and protein expression profiles of Chinese hamster ovary (CHO) clones producing an tsAb were analyzed in the exponential growth and stationary (tsAb production) phase of fed-batch culture. Functional analysis revealed activated endoplasmic reticulum stress in high mispairing clones in both culture phases, while low mispairing clones exhibited expression profiles indicative of activated protein translation, as well as higher endocytosis and target protein degradation, suggesting the clearance of unfolded proteins through ubiquitin-mediated mechanisms. In addition, through transcriptomic profiling, we identified a group of genes that have the potential to be used as a biomarker panel tool for identifying high mispairing levels in the early stages of bioprocess development.