Chinese hamster ovary cells continuously secrete a cysteine endopeptidase

Abstract

The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide, was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium. In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases.

Mapping and partial characterization of proteases expressed by a CHO production cell line

“…Thus, the metalloproteinase(s) are assumed to be responsible for the greater part of the protease activity liberated by the CHO cells. A smaller portion of the protease activity was due to the presence of serine and cystein proteases, which has been described previously in the supernatant of CHO cells (Satoh et al, 1990). Further evidence for the presence of cystein proteases, as measured in the cell supernatant, was seen by the significant cleavage occurring with peptide substrates Z-Phe-Arg-MCA and Boc-Leu-Arg-Arg-MCA, which were previously reported to be sensitive to cleavage by cystein proteases (Satoh et al, 1990).…”

“…A smaller portion of the protease activity was due to the presence of serine and cystein proteases, which has been described previously in the supernatant of CHO cells (Satoh et al, 1990). Further evidence for the presence of cystein proteases, as measured in the cell supernatant, was seen by the significant cleavage occurring with peptide substrates Z-Phe-Arg-MCA and Boc-Leu-Arg-Arg-MCA, which were previously reported to be sensitive to cleavage by cystein proteases (Satoh et al, 1990). Fluorescence (absorbance at 380 nm and emission at 460 nm), measured after incubation with the two substrates for 22 h at 378C, showed a 10-fold and a 15-fold increase in fluorescence over the controls.…”

Mammalian cell-produced therapeutic proteins: heterogeneity derived from protein degradation

“…For BHK cell cultures, a dipeptidyl-aminopeptidase has been described by Gawlitzek et al [60]. Satoh et al [61] and Sandberg et al [38] described the production of two types of proteases from CHO cultures -exopeptidases and endopeptidases. The exopeptidase had an aminopeptidase activity which increased linearly with culture time and correlated with an increase in the non-viable cell count, suggesting its release from lyzed cells.…”

Aqueous Stability of Recombinant Human Thrombopoietin as a Function of Processing Schemes

“…[15][16][17] Yet, proteolytic problems during bioprocessing and purification do not always receive detailed coverage in the literature, 18 especially with regard to the discovery and development of pharmaceutical proteins. As evident by these studies with rhTPO, distinctions between degradation characteristics of purification variants can be a function of the "quality" of the preparations, and the optimal composition and storage conditions (pH, temperature, etc.)…”