Structural characterization of heparins from different commercial sources
- 416 Downloads
Seven commercial heparin active pharmaceutical ingredients and one commercial low molecular weight from different manufacturers were characterized with a view profiling their physicochemical properties. All heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis (M N, 10–11 kDa; M W, 13–14 kDa; polydispersity (PD), 1.3–1.4) and by size exclusion chromatography (M N, 14–16 kDa; M W, 21–25 kDa; PD, 1.4–1.6). one-dimensional 1H- and 13C-nuclear magnetic resonance (NMR) evaluation of the heparin samples was performed, and peaks were fully assigned using two-dimensional NMR. The percentage of glucosamine residues with 3-O-sulfo groups and the percentage of N-sulfo groups and N-acetyl groups ranged from 5.8–7.9%, 78–82%, to 13–14%, respectively. There was substantial variability observed in the disaccharide composition, as determined by high performance liquid chromatography (HPLC)-mass spectral analysis of heparin lyase I–III digested heparins. Heparin oligosaccharide mapping was performed using HPLC following separate treatments with heparin lyase I, II, and III. These maps were useful in qualitatively and quantitatively identifying structural differences between these heparins. The binding affinities of these heparins to antithrombin III and thrombin were evaluated by using a surface plasmon resonance competitive binding assay. This study provides the physicochemical and activity characterization necessary for the appropriate design and synthesis of a generic bioengineered heparin.
KeywordsHeparin Polyacrylamide gel electrophoresis Size exclusion chromatography analysis Molecular weight properties Disaccharide composition High performance liquid chromatography–mass spectrometry Oligosaccharide mapping Nuclear magnetic resonance spectroscopy Surface plasmon resonance
This work was supported by grants funded by the National Institutes of Health HL101721 and HL096972 (RJL) and the Bioengineered Heparin Consortium.
- 4.Nader HB, Dietrich CP (1989) Natural occurrence and possible biological role of heparin. In: Lane DA, Lindahl U (eds) Heparin chemical and biological properties, clinical applications. CRC, Boca Raton, pp 115–133Google Scholar
- 8.Guerrini M, Beccati D, Shriver Z, Naggi AM, Bisio A, Capila I, Lansing J, Guglieri S, Fraser B, Al-Hakim A, Gunay S, Viswanathan K, Zhang Z, Robinson L, Venkataraman G, Buhse L, Nasr M, Woodcock J, Langer R, Linhardt RJ, Casu B, Torri G, Sasisekharan R (2008) Oversulfated chondroitin sulfate is a major contaminant in heparin associated with adverse clinical events. Nat Biotechnol 26(6):669–775CrossRefGoogle Scholar
- 19.Zhang Z, Xie J, Liu H, Liu J, Linhardt RJ (2009) Quantitification of heparan sulfate and heparin disaccharides using ion pairing, reverse-phase, micro-flow, high performance liquid chromatography coupled with electrospray ionization trap mass spectrometry. Anal Chem 81(11):4349–4355CrossRefGoogle Scholar
- 26.Linhardt RJ (1994) In: Varki A (ed) Current protocols in molecular biology: analysis of glycoconjugates. Wiley-Interscience, Hoboken, pp 17.13.17–17.13.32Google Scholar
- 27.Merchant ZM, Kim YS, Rice KG, Linhardt RJ (1985) Structure of heparin-derived tetrasaccharides. Biochem J 229(2):369–377Google Scholar
- 30.Linhardt RJ, Kerns RJ, Vlahov IR (1996) In: Yalpani M (ed) Heparin and heparin oligosaccharides: preparation, analysis, applications and biological activities, biochemical functions and biotechnology of natural and artificial polymers. ATL Press, Science Publishers, Mt. Prospect, pp 46–62Google Scholar
- 31.Linhardt RJ (1991) Heparin: an important drug enters its seventh decade. Chem Ind 2:45–50Google Scholar
- 32.Wang Z, Ly M, Zhang F, Zhong W, Suen A, Dordick JS, Linhardt RJ (2010) E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor. Biotechnol Bioengin 107(6):968–977Google Scholar