Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity

Munzert E, Müthing J, Büntemeyer H, Lehmann J (1996)
BIOTECHNOLOGY PROGRESS 12(4): 559-563.

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Abstract
Sialidase activity in cell-free supernatant of batch-cultivated Chinese hamster ovary (CHO) cells producing human recombinant antithrombin III (rhAT III) was monitored during cultivation using 4-methylumbelliferyl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increasing sialidase activity which seemed to be principally due to cell lysis, resulting in release of cytosolic sialidase. Loss of terminally alpha(2-->3) bound sialic acids of the oligosaccharides of rhAT III was analyzed in lectin-based Western blot and enzyme-linked lectin assays, using Maackia amurensis and Datura stramonium agglutinins for specific determination of Neu5Ac alpha(2-->3)Gal- and Gal beta(1-->4)GlcNAc-terminated glycoproteins, respectively. Results show a remarkable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells throughout long-term batch cultivation. To avoid this degradation effect, process parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal recombinant protein concentrations is highly recommended to avoid product desialylation.
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Munzert E, Müthing J, Büntemeyer H, Lehmann J. Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity. BIOTECHNOLOGY PROGRESS. 1996;12(4):559-563.
Munzert, E., Müthing, J., Büntemeyer, H., & Lehmann, J. (1996). Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity. BIOTECHNOLOGY PROGRESS, 12(4), 559-563.
Munzert, E., Müthing, J., Büntemeyer, H., and Lehmann, J. (1996). Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity. BIOTECHNOLOGY PROGRESS 12, 559-563.
Munzert, E., et al., 1996. Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity. BIOTECHNOLOGY PROGRESS, 12(4), p 559-563.
E. Munzert, et al., “Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity”, BIOTECHNOLOGY PROGRESS, vol. 12, 1996, pp. 559-563.
Munzert, E., Müthing, J., Büntemeyer, H., Lehmann, J.: Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity. BIOTECHNOLOGY PROGRESS. 12, 559-563 (1996).
Munzert, E, Müthing, Johannes, Büntemeyer, Heino, and Lehmann, J. “Sialidase activity in culture fluid of Chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity”. BIOTECHNOLOGY PROGRESS 12.4 (1996): 559-563.
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