Monitoring intracellular protein degradation in antibody-producing Chinese hamster ovary cells

Intracellular proteolysis in mammalian cells is a native cellular strategy to recycle proteins and peptides. Whether or not this mechanism may hamper monoclonal antibody (mAb) formation in Chinese hamster ovary (CHO) cells was the key driver for this study. Exponentially growing, anti-interleukin (IL)-8 producing CHO cells were fed with C-13-labeled L-lysine. The fate of the labeling signal was tracked in intracellular peptides, which were the proteolytic fragments of the mAb. Signal analysis was performed in samples after cell disruption, anion exchange SPE and Q-ToF mass detection. Four degradation peptides were found, with HYTQKSLSLSPGK and HYTQKSLSLSPG containing two and one L-lysine unit (K), respectively. Labeling dynamics were used for model-based identification of the degradation rate in four biological replicates. Degradation rates of 22-25 pmol/10(8)cells/h were estimated, representing about 3% of the net mAb production. Hence, intracellular mAb degradation occurs even under the rather smooth production conditions installed.