Abstract
The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.
Highlights
From the $Department of Biochemistry, Institute of Medical Science, Universiotyf Tokyo, Minato-ku, Tokyo108, Japun, the $Pharmacology Institute, Bayer Yakuhin Ltd., Kobe651-23, Japan, the WutterBiological, Miles Znc., Berkeley, California 94701, and the IlMedicine and Development Division, Bayer Yakuhin, Ltd., Chuo-ku, Osaka541, Japan
Extensive biochemical characterization of rFVIII synthesized in BHK cells indicated serial chromatography onimmobilized lectin columns that it is similar to human plasma-derived factor VI11
Like pdFVIII, rFVIII is proteolytically cleaved at oligosaccharide by sequential exo- and endoglycosi- amino acid 1648 to generate a 200-kDa N-terminal fragment dase digestion and by methylation analysis revealed and an 80-kDa C-terminal fragment that remain associated that both factorVI11 preparations contain mainly high in the presence of divalent cations
Summary
It has been reported that recombinant glycoproteins produced by different cell lines often have different oligosaccharide structures [36,37,38,39]. pdFVIII wasmade in human cells, most likely in the liver parenchymal cells [3], while rFVIII which has a greater proportion of a-galactose residues. Additional data supporting these obreported in this paper, high mannose-type, and bi-, tri-, and servations were obtained from clinical studies in which the tetra-antennary complex-type sugar chains were found in recoveries and half-lives of pdFVIII and rFVIII infused into both pdFVIII and rFVIII (Table 111).These data accord with the hemophilic patients were determined. One of the All ofthese in uiuo studies indicated that a-galactose residues major differences is that a portion of the biantennary com- do not affect rFVIII pharmacokinetic parameters in baboons plex-type sugar chains of pdFVIII contains the blood group A and patientswith hemophilia. Was detected in the sugar chains of rFVIII-E than inthose of rFVIII, derived from a different transfected BHK cell isolate
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