Structural variation in the linkage region of pharmaceutical heparin arising from oxidative treatments during manufacture

Abstract

During the manufacture of pharmaceutical heparin, a range of treatments are applied to sanitize, decolourise and reduce the pyrogenic properties of the samples. The structural effects of bleaching, an oxidative process, are examined. Among ¹H and ¹³C NMR signals ascribable to the tetrasaccharide linkage region of heparin, samples of porcine mucosal heparin frequently display characteristic signals at chemical shift values of 4.5 and 106 ppm respectively, which have not been explained previously. Fractions enriched with material reporting this signal were isolated from heparinase digested porcine mucosal heparin samples and subjected to analysis using mass spectrometry and NMR spectroscopy. A novel structure, ΔU-Gal-Gal-Xyl-CH₂-CONH₂, was identified by mass fragmentation experiments and further interesting structural motifs emerged following evaluation by mass spectrometry of longer oligosaccharide chains biosynthesized away from the linker tetrasaccharide, GlcA-Gal-Gal-Xyl. The carbohydrate-protein linkage region is thus affected by the bleaching step involved in the manufacturing process of heparin. The discovery of specific modifications that reflect the extent of the oxidation treatment adopted is relevant to the monitoring of inadvertent damage to the heparin structure during manufacture that contributes to sample variation and which could also lead to reduced drug quality.

Keywords: Heparin linkage region; Heparin lyase; Mass spectrometry; Mucosal heparins.