IMPORTANCE OF SIALIC-ACID IN RECOMBINANT THROMBOMODULIN IN TERMS OF PHARMACOKINETICS AND SEPARATION OF DESIALYZED GLYCOPROTEIN

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM ) expressed in mouse C-127 cells has potent antithrombotic activity av ailable as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asial o GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltra nsferase, containing sialic acid similarly to native sialo GAG-UTM, ha d only a short half-life in plasma, suggesting that the binding site o f sialic acid on galactose was not only sialyzed with alpha-2, 6-sialy ltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however had a long half-life. These resu lts suggest that terminal sialic acid may be important to the pharmaco kinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became s ignificant for quality control. In order to analyze sialo- and asialo- types in the early stage of purification, we investigated separation a nd analysis methods for both types and found a suitable sample of each : RCA-120-Agarose column for separation and ELISA using anti-thrombomo dulin antibody and RCA lectin for analysis.