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Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes. [ABSTRACT FROM AUTHOR]