PRODUCTION OF RECOMBINANT HUMAN ANTITHROMBIN-III ON 20-L BIOREACTOR SCALE - CORRELATION OF SUPERNATANT NEURAMINIDASE ACTIVITY, DESIALYLATION, AND DECREASE OF BIOLOGICAL-ACTIVITY OF RECOMBINANT GLYCOPROTEIN

Chinese hamster ovary (CHO) cells producing the recombinant glycoprote in human antithrombin III (rhAT III) were batch cultivated in a 20-L b ioreactor for 13 days. Neuraminidase activity in cell-free supernatant was monitored during cultivation and free sialic acid was determined by HPLC. Neu5Ac alpha(2-->3)Gal-specific Maackia amurensis and Gal bet a(1-->4)GlcNAc-specific Datura stramonium agglutinin were used for det ermination of sialylated and desialylated rhAT III, respectively. A co mmercial test kit was used for evaluation of functional rhAT III activ ity. Supernatant neuraminidase as well as lactate dehydrogenase activi ty increased significantly during batch growth. The enhanced number of dead cells correlated with increased neuraminidase activity, which se emed to be principally due to cell lysis, resulting in release of cyto solic neuraminidase. Loss of terminally alpha(2-->3) linked sialic aci ds of the oligosaccharide portions of rhAT III, analyzed in lectin-bas ed Western blot and lectin-adsorbent assays, correlated with a decreas e of activity of rhAT III produced throughout long-term batch cultivat ion. Thus, structural oligosaccharide integrity as well as the functio nal activity of recombinant glycoprotein depend on the viability and m ortality of the bioreactor culture, and batches with a high number of viable cells are required to guarantee production of glycoproteins wit h maximum biological activity. (C) 1997 John Wiley & Sons, Inc.