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 available 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. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with alpha-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo- and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each: RCA-120-Agarose column for separation and ELISA using anti-thrombomodulin antibody and RCA lectin for analysis.
Edano
T
T
Tokyo Research Laboratories, Kowa Co., Ltd., Higashimurayama, Japan.
Koshi
T
T
Yoshizaki
H
H
Kumai
N
N
Habata
Y
Y
Koide
T
T
Mizoguchi
T
T
Yamamoto
S
S
Kimata
H
H
Ohkuchi
M
M
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007275:Injections, Intravenous; D008297:Male; D051379:Mice; D011786:Quality Control; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D012794:Sialic Acids / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology*; D018180:Thrombomodulin / Q000737:chemistry*
D000925:Anticoagulants; D006023:Glycoproteins; D011994:Recombinant Proteins; D012794:Sialic Acids; D018180:Thrombomodulin