Protease activity in protein-free NSO myeloma cell cultures

Abstract

Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.

Suppression of IgM Proteolysis by Conformational Stabilization Through Excipients

“…Host cell proteases may cause significant product loss or affect the quality of recombinant proteins [ 1 ]. Proteases are found in several host cells like cathepsin D-like proteases in hybridoma cells [ 1 ], aspartic acid and cysteine proteases like cathepsin L in myeloma cells [ 2 ], gelatinase B and cathepsin D in Chinese hamster ovary (CHO) cells [ 3 , 4 ], or serine and cysteine proteinases in the plant, Nicotiana benthamiana [ 5 ].…”