Cell lines for industrial pharmaceutical protein production processes need to be robust, fast-growing, and high-producing. In order to find such cells, we performed a high passage cultivation of monoclonal antibody producing Chinese hamster ovary (CHO) cells in shaking flasks for more than 420 days. Examinations of cell growth, productivity, intracellular protein, and metabolite characteristics as well as product transcript and genomic integrate levels revealed substantial differences between subpopulations that were cryopreserved from long-term cultivation at different time points. Detected growth performance as well as intracellular adenylate energy charge increased during high passage cultivation. In addition, proteome analysis indicated an augmented utilization of glycolysis with higher passage number and an enhanced robustness based on anti-stress proteins. Interestingly, the product formation increased at first but decreased dramatically during the later subcultivations, although selection pressure was applied. Utilizing flow cytometry and quantitative real-time polymerase chain reaction, we further examined the translational, transcriptional, and genomic basis for the observed phenotypes. The detected reduction of antibody expression, in particular of the heavy chain, was ascribed to a decrease of antibody transcript, caused by loss of gene copy number and assumably a malfunctioning splicing mechanism of the dicistronic mRNA. To our knowledge, this is the first systematic approach using process analytics and targeted omic techniques to elucidate the effects of long-term cultivation of CHO cells expressing a therapeutic protein.