A novel acid proteinase relased by hybridoma cells

Abstract

An acid proteinase has been detected in culture supernate of the 9.2.27 murine hybridoma. This enzyme extensively degrades albumin and transferrin during short incubations at pH 3 and below. Limited proteolysis of the 9.2.27 IgG2a appears to occur in the culture supernate. Proteolysis in enhanced at low pH in the presence of urea or 1 M acetic acid. The proteinase activity accumulates in continuous perfusion, total cell recycle cultures, beginning during exponential growth of the hybridoma. It is destroyed by boiling and blocked by pepstatin, but not by inhibitors of cysteine or serine proteinases or by EDTA. The low pH optimum may distinguish this enzyme from the known rat and mouse aspartic acid proteinases including cathepsin D and cathepsin E.

Protease Activity in Protein-Free Ns0 Myeloma Cell Cultures

“…1 and 2). Aspartic acid protease activity has also been found in hybridoma cell cultures (Schlaeger et al, 1987;Karl et al, 1990;Van Erp et al, 1991). Two activities were identified as a cathepsin D-like enzyme having a momomeric and a homodimeric form at around 45 and 90 kDa.…”

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…It is likely that the difference in viability observed after 16 days in perfusion culture between the NS/0 control (25%) and E1B-19K cells (55%) would have been even greater if the perfusion cultures had been prolonged further. One clear benefit of this increased viability is a substantial reduction of the cell-derived components known to be responsible for degradation of the product and complications in cell/medium separation and downstream processing (Karl et al, 1990;Maiorella et al, 1991;Van Erp et al, 1991).…”

“…Furthermore, increased levels of cellular-derived substances such as DNA are prone to complicate cell/medium separations (Maiorella et al, 1991). The release of proteases and other intracellular enzymes can result in oxidation, deamination and oligosaccharide hydrolysis of the proteins of interest (Karl et al, 1990;Mohan et al, 1993;Van Erp et al, 1991) with deleterious consequences such as enhanced clearance rates of injected products in therapeutic applications (Goochee and Monica, 1990;Maiorella et al, 1993;Prior et al, 1989).…”

Characterization of the proteases involved in the N‐terminal clipping of glucagon‐like‐peptide‐1‐antibody fusion proteins

“…Interestingly, although the metalo‐proteases were determined to be the major proteases in the CHOK1 culture fluid (Elliott P. et al,2003; Sandberg et al,2006) and an aspartic acid protease was determined to be responsible for the degradation of Fc‐fusion protein in CHO‐DUKX‐B11 (Flavie et al,2009), the protease make‐up of CHOK1SV is unknown (but see below). Other strains of CHO cell lines are known to produce protease (Elliott P. et al,2003; Mols et al,2005; Sandberg et al,2006; Satoh et al,1990) as are also hybridoma cells (Karl et al,1990; Van Erp et al,1991).…”