Structural Characterization of Pharmaceutical Heparins Prepared from Different Animal Tissues

Abstract

Although most pharmaceutical heparin used today is obtained from porcine intestine, heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of heparin. This concern began with the outbreak of mad cow disease in the 1990s and was exacerbated during the heparin shortage in the 2000s and the heparin contamination crisis of 2007–2008. Three heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin III-binding affinity were observed. These data provide some insight into the variability of heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a heparin active pharmaceutical ingredient.

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

“…Pharmaceutical heparin characteristically has a varied disaccharide composition, composed of seven to eight different disaccharides and dominated by a preponderance of TriS (43,44). In contrast, CHO-S cells produce HS with a simpler structure, primarily composed of the N-acetylated 0S disaccharide with smaller amounts of NS as well as minor amounts of disaccharides containing O-sulfo groups.…”

“…When pharmaceutical heparins are treated with heparin lyase-2, as many as five tetrasaccharides are formed, with 3-Osulfoglucosamine at their reducing ends (50). These correspond to a structural signature of the AT-binding site structural variants correlating with the anticoagulant activity of heparin (44). A tetrasaccharide analysis was performed on heparin lyase-2-treated HS from CHO-S, Dual-29, Dual-10, and CHO-gt31 cells and spent media.…”

“…7) and spent media (0.6%) afforded HS with a heparin lyase-2-resistant tetrasaccharide of the structure ⌬UA-GlcNAc6S-GlcUA-GlcNS3S, consistent with the action of HS3st1 creating an AT-binding site in HS from CHO-gt31 cells (Table 3). Based on biochemical studies and the crystal structure of the ternary complex of HS3st1 with PAPS and an HS oligosaccharide, HS3st1 has a known substrate specificity that indicates that it can act on GlcN(Ac/ S)6(S/OH)-GlcUA-GlcNS3OH(6S/OH)-IdoUA2S-GlcNS(6S/OH) (51), resulting in as many as the five structural variants of ATbinding sites observed in pharmaceutical heparins (44). Although CHO-gt31 cells and spent media contain an HS with a GlcNAc6S-GlcUA-GlcNS3S6S-IdoUA2S-GlcNS(6S/OH) ATbinding site, these cells have only exogenous HS3st1; endogenous levels of NDST1, HS2st, GLCE, and HS6sts; and no detectable levels of NDST2 (38,39).…”

“…The activation of AT by heparin relies on a bridging mechanism for thrombin and is sensitive only to conformational change (allosteric) for factors Xa and IXa. Pharmaceutical heparin from different sources has differential amounts of AT-binding sites and exhibits some differences in anti-factor Xa activity (44). The highly N-sulfated HS from Dual-29 cells does not contain any 3-O-sulfo groups as determined by tetrasaccharide analysis and thus does not contain AT-binding sites and shows a relatively low anti-factor Xa activity (2-4 units/mg) (39).…”

“…Fraction 1 had an antifactor Xa activity of 0.6 Ϯ 0.5 units/mg, whereas Fraction 2 showed an anti-factor Xa activity of 137 Ϯ 36 units/mg. The anti-factor Xa activity of pharmaceutical heparin is Ͼ180 units/mg (44).…”