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Abstract A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high‐producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three‐level factorial experimental design was performed in fed‐batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter‐influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late‐stage ROS activity in a dose‐dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity. 

Abstract Trace metals are supplied to chemically‐defined media (CDM) for optimal Chinese hamster ovary (CHO) cell culture performance during the production of monoclonal antibodies and other therapeutic proteins. However, lot‐to‐lot and vendor‐to‐vendor variability in raw materials consequently leads to an imbalance of trace metals that are supplied to CDM. This imbalance can yield detrimental effects rooted in several primary mechanisms and pathways including oxidative stress, apoptosis, lactate accumulation, and unfavorable glycan synthesis. Recent research endeavors involve supplying zinc, copper, and manganese to CDM in excess to further maximize culture productivity and product quality. These treatments significantly impact critical quality attributes and furthermore highlight the degree to which trace metal availability can affect CHO cell culture performance. This review highlights the role of trace metal variability, supplementation, and interplay on key cellular mechanisms responsible for overall culture performance and the production and quality of therapeutic proteins.