Evaluation of the proteolytic potential of in vitro-cultivated hybridoma and recombinant mammalian cells

Abstract

The proteolytic potential of culture supernatants derived from recombinant baby hamster kidney (BHK) 21 and mouse-mouse hybridoma cells have been characterized. Several assays using enzyme specific chromogenic artificial peptides, as well as a radioactive test for the detection of the total activity, have been established and were adapted to the special conditions existing in culture media of mammalian cells. Proteolytic activity was detected in human serum albumin containing media which was specific for peptides ending with a terminal arginine. The addition of N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid (HEPES) buffer to the culture media resulted in a significant peptide cleavage potential, supporting the fact that this compound is not recommended as a supplement in animal cell culture media. Medium shock protease activity has been detected in culture supernatants of BHK cells when medium was changed completely, caused by a switch from a serum containing state of growth to a serum-free state of growth which is often used in processes with microcarriers. However, this proteolytic activity showed a transient behaviour whereby its secretion stopped when the cells had adapted to the serum-free medium conditions. Characterization of the proteolytic activities using different specific inhibitors and activators supported the assumption that the proteolytic activity reflects a cell specific composition of proteases which can also change dependent on the culture conditions used.