Effects of Extracellular Enzymes

Extracellular enzymes, namely proteases and glycosidases, are believed to play an important role in protein glycosylation (8,109). A properly glycosylated recombinant protein is thought to be more resistant to protease degradation (18). Since media protein components are also substrates for extracellular proteases, industry's switch to serum-free and protein-free medias could potentially increase the probability of proteolytic degradation of the recombinant glycoprotein.

There are five identified types of glycosidases: sialidases, b-galactosidases, b-hexosaminidases, mannosidases, and fucosidases (109). Glycosidases that are most important to recombinant protein production are: sialidases and b-galactosidases. Sialidases remove terminal sialic acids and b-galactosidases remove terminal galactoses. Both terminal sialic acid and galactose increase the half-life of a recombinant protein in circulation. For a glycosidase to cleave sugars from the glycoprotein, the glycosidase needs to be expressed in the cell, secreted into the media, have specificity to the glycoprotein, and be stable in the media. Therefore, culture conditions that minimize extracellular glycosidases will result in increased uniformity and stability of the recombinant glycoprotein (109).

Goochee and Gramer (110,111) characterized glycosidases from CHO, adeno-virus-transformed human embryonic kidney (293), mouse myeloma (NS0), and a hybridoma cell line. The glycosidases isolated from the CHO cell supernatant were very stable over a wide range of pH and temperature. The glycosidases isolated from 293, NS0, and the hybridoma cell line supernatants were not very stable at neutral pH or 37° C. The glycosidase activity in CHO cells increased when the cells were damaged (112). Munzert et al. (113) reported both sialidase activity and loss of protein sialylation. Ferrari et al. (114) used antisense technology to reduce the sialidase expression in CHO producing DNase. The final product had a 20-30% increase in sialylation (114).

Kratje et al. (115) conducted a comprehensive study of CHO proteases. They determined that one of the main issues with serum-free cultivations is proteolytic attack and degradation of the recombinant protein. They observed that the cell-specific protease composition changed, depending on the culture environment. Protease activity was observed to spike after a media exchange. Normal protease levels returned after the cells had adapted to the fresh media. Yang and Butler (60) also noted increased protease activity in serum-free media, observed as truncated forms of EPO. They hypothesized that amino acid starvation might have caused the elevated protease activity. Satoh et al. (116) first reported two types of proteases in CHO, an endoprotease and an exopeptidase. The exopeptidase activity increased with culture time and correlated with the nonviable cell count. Endo-protease activity was observed to have cysteine protease characteristics and was secreted continuously by viable cells. No molecular weights were reported for these proteases. Froud et al. (117) observed extracellular proteases in CHO that were upre-gulated during amino acid starvation. Cartwright (118) stated that protease activity was directly due to amino acid starvation in CHO cells. Gawlitzek et al. (75) reported that eight amino acids were removed from IL-2 expressed in BHK cells. Goldman et al. (119) reported up to 10 amino acids removed from IFN-g. Wang et al. (85) reported EPO degradation by proteases. EPO contained a protease-sensitive region that bisected the molecule. Therefore, protease and glycosidase activity should be assessed and monitored during the production of recombinant glycoproteins.

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Responses

  • gisella palermo
    How to remove protease activity in cho cells?
    7 years ago

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