From Discovery to Delivery: A Rapid and Targeted Proteomics Workflow for Monitoring Chinese Hamster Ovary Biomanufacturing.

Chinese hamster ovary (CHO) cells are the industrial workhorse for manufacturing biopharmaceuticals, including monoclonal antibodies. CHO cell line development requires a more data-driven approach for the accelerated identification of hyperproductive cell lines. Traditional methods, which rely on time-consuming hierarchical screening, often fail to elucidate the underlying cellular mechanisms driving optimal bioreactor performance. Big data analytics, coupled with advancements in "omics" technologies, are revolutionizing the study of industrial cell lines. Translating this knowledge into practical methods widely utilized in industrial biomanufacturing remains a significant challenge. This study leverages discovery proteomics to characterize dynamic changes within the CHO cell proteome during a 14-day fed-batch bioreactor cultivation. Utilizing a global untargeted proteomics workflow on both a ZenoTOF 7600 and a Cyclic IMS QToF, we identify 3358 proteins and present a comprehensive data set that describes the molecular changes that occur within a well-characterized host chassis. By mapping relative abundances to key cellular processes, eight protein targets were selected as potential biomarkers. The abundance of these proteins through the production run is quantified using a 15-min targeted triple quadrupole (MRM) assay, which provides a molecular-level QC for cell viability. This discovery to target workflow has the potential to assist engineering of new chassis and provide simple readouts of successful bioreactor batches.