SIALIDASE ACTIVITY IN CULTURE FLUID OF CHINESE-HAMSTER OVARY CELLS DURING BATCH CULTURE AND ITS EFFECT ON RECOMBINANT HUMAN ANTITHROMBIN-III INTEGRITY

Sialidase activity in cell-free supernatant of batch-cultivated Chines e hamster ovary (CHO) cells producing human recombinant antithrombin I II (rhAT III) was monitored during cultivation using 4-methylumbellife ryl substrate and HPLC for free sialic acid determination. Supernatant sialidase as well as lactate dehydrogenase activity increased signifi cantly during batch growth. The enhanced number of dead cells correlat ed with increasing sialidase activity which seemed to be principally d ue 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 agglutini ns for specific determination of Neu5Ac alpha(2-->3)Gal- and Gal beta( 1-->4)GlcNAc-terminated glycoproteins, respectively. Results show a re markable loss of terminal sialic acids of rhAT III along with decrease in CHO cell viability and concomitant increase of dead cells througho ut long-term batch cultivation. To avoid this degradation effect, proc ess parameters forcing high viability are essential and harvesting of culture at an early time even at suboptimal recombinant protein concen trations is highly recommended to avoid product desialylation.