A review.The idea of making transgenic drugs from mammary glands of goats is discussed.The European Medicines Evaluation Agency (EMEA) may decide early next year on approval of an anticoagulant protein, human antithrombin, that is produced in goat's milk to treat hereditary disorder.If the drug, ATryn, finally gest a nod from regulators, its approval will mark the culmination of a meandering 15-yr journey for GTC biotherapeutics, a Framingham, Mass., spin-off of the biotech gian Genzyme.

1997-11-20·Biotechnology and Bioengineering2区 · 工程技术

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

2区 · 工程技术

Article

作者: Munzert, Eberhard ; Lehmann, Jurgen ; Buntemeyer, Heino ; Heidemann, Rudiger ; Muthing, Johannes

Chinese hamster ovary (CHO) cells producing the recombinant glycoprotein human antithrombin III (rhAT III) were batch cultivated in a 20-L bioreactor for 13 days. Neuraminidase activity in cell-free supernatant was monitored during cultivation and free sialic acid was determined by HPLC. Neu5Acalpha(2-->3)Gal-specific Maackia amurensis and Galbeta(1-->4)GlcNAc-specific Datura stramonium agglutinin were used for determination of sialylated and desialylated rhAT III, respectively. A commercial test kit was used for evaluation of functional rhAT III activity. Supernatant neuraminidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increased neuraminidase activity, which seemed to be principally due to cell lysis, resulting in release of cytosolic neuraminidase. Loss of terminally alpha(2-->3) linked sialic acids of the oligosaccharide portions of rhAT III, analyzed in lectin-based Western blot and lectin-adsorbent assays, correlated with a decrease of activity of rhAT III produced throughout long-term batch cultivation. Thus, structural oligosaccharide integrity as well as the functional activity of recombinant glycoprotein depend on the viability and mortality of the bioreactor culture, and batches with a high number of viable cells are required to guarantee production of glycoproteins with maximum biological activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 441-448, 1997.

1985-10-01·Seminars in Thrombosis and Hemostasis2区 · 医学

Human Antithrombin Concentrates and Experimental Disseminated Intravascular Coagulation

2区 · 医学

Article

作者: Brown, Janet M. ; Mammen, Eberhard F. ; Miyakawa, Teruo ; Murano, Genesio ; Assarian, Gary S. ; Phillips, Thomas F.

The effect of human antithrombin III concentrates was studied in a dog model in which DIC was produced by the infusion of 0.66 M lactic acid. In 35 animals infused with lactic acid only, platelet counts, fibrinogen levels, alpha 2-antiplasmin levels, and antithrombin levels decreased significantly over a 4-hour observation period. At the same time, the levels of fibrin(ogen) split products increased. At the end of the experimental period, the glomeruli of the dogs' kidneys contained significant quantities of fibrin. Hemodynamically, the dogs experienced a significant decrease in cardiac output, whereas the mean arterial blood pressures remained unchanged. Urine output seemed to decline during the time of study. Appreciable quantities of heparin could be measured in the dogs' plasmas. In 13 dogs that were treated in the same manner as the just described 35 animals, 1 U of human antithrombin concentrate per ml of calculated dog plasma was infused over a 90-minute time span, beginning after 1 hour of acidosis. Although no differences were noted in the decrease in platelet counts between the two groups, fibrinogen levels and alpha 2-antiplasmin levels declined less drastically in the antithrombin-treated group. Also the levels of fibrin(ogen) split products increased less in the treated group. The antithrombin activity levels increased markedly and remained at their high levels even after the infusion of antithrombin had ceased. The amount of human antithrombin circulating was followed by immunologic assays using a human antibody to antithrombin. Also, with this technique, the levels of infused antithrombin did not decrease. The amount of fibrin found in the glomeruli of this group of animals was significantly lower than in the nontreated group (p less than 0.001). Even cardiac output was higher in the treated group and urine production seemed to increase. Also, these dogs had appreciable quantities of heparin in their plasmas. In a control group of six animals who were infused with saline instead of lactic acid, no major changes were observed in the coagulation and cardiovascular parameters. These data seem to suggest that antithrombin concentrates exert a certain protective effect in this particular DIC animal model.

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