CHO cell lines growing in suspension were cultivated at 36.5 C° and 10% CO2 in shake flasks using a proprietary, chemically defined culture medium. Gene editing was performed according to manufacturer's protocols, cells were single cell sorted using FACS device and subsequently screened for the desired phenotype. Cell viabilities and growth rates were monitored using an automated system (ViCell, Beckman Coulter). Cells were stably transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany) with expression plasmids encoding human monoclonal antibodies. This and all other kits were used according to the manufacturer's instructions.
Spike-in experiments were performed in conditioned medium. Conditioned medium was collected by centrifugation of the cells grown for 7/8 days for 15 min at 90 g. After centrifugation, the supernatant was transferred and passed through a 0.22 μm filter to remove remaining cell particles from the conditioned medium. Under these conditions and at this stage of cell growth, the maximum amounts of secreted proteases are expected to be active in the cell culture medium without release of intracellular proteases due to cell death. The polypeptide of interest was added to the conditioned medium with a final concentration of 0.7 μM and incubated at 37°C with continuous shaking at 500 rpm. After incubation, samples were analyzed by SDS-PAGE followed by Western Blot analysis to determine the amount of clipping.