Abstract
Danaparoid sodium salt, is the active component of ORGARAN, an anticoagulant and antithrombotic drug constituted of three glycosaminoglycans (GAGs) obtained from porcine intestinal mucosa extracts. Heparan sulfate is the major component, dermatan sulfate and chondroitin sulfate being the minor ones. Currently dermatan sulfate and chondroitin sulfate are quantified by UV detection of their unsaturated disaccharides obtained by enzymatic depolymerization. Due to the complexity of danaparoid biopolymers and the presence of shared components, an orthogonal approach has been applied using more advanced tools and methods. To integrate the analytical profile, 2D heteronuclear single quantum coherence (HSQC) NMR spectroscopy was applied and found effective to identify and quantify GAG component signals as well as those of some process signatures of danaparoid active pharmaceutical ingredient (API) batches. Analyses of components of both API samples and size separated fractions proceeded through the determination and distribution of the molecular weight (Mw) by high performance size exclusion chromatographic triple detector array (HP-SEC-TDA), chain mapping by LC/MS, and mono- (1H and 13C) and bi-dimensional (HSQC) NMR spectroscopy. Finally, large scale chromatographic isolation and depolymerization of each GAG followed by LC/MS and 2D-NMR analysis, allowed the sequences to be defined and components to be evaluated of each GAG including oxidized residues of hexosamines and uronic acids at the reducing ends.
Highlights
Danaparoid sodium, constituted by a mixture of Low Molecular Weight (LMW) heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS), extracted from porcine intestinal mucosa, is the active component of ORGARAN an anticoagulant and antithrombotic drug approved for prophylaxis of post-operative deep-vein thrombosis
Analysis of molecular weight distribution was performed on seven active pharmaceutical ingredient (API) samples of danaparoid using high performance size-exclusion chromatography (HP-size exclusion chromatography (SEC)) on polymeric columns, combined with a triple detector array (TDA) and UV detector
The in depth studies conducted on SEC fractions and on isolated CS/DS and HS components produced much structural information that completed the characterization of the starting API samples and allowed the similarity of analyzed batches to be established, ascribing slight differences to the intrinsic heterogeneity of extractive products such as danaparoid
Summary
Danaparoid sodium, constituted by a mixture of Low Molecular Weight (LMW) heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS), extracted from porcine intestinal mucosa, is the active component of ORGARAN an anticoagulant and antithrombotic drug approved for prophylaxis of post-operative deep-vein thrombosis. Alternative more feasible NMR quantifications have been developed for the components of danaparoid API batches [5] and for heparin composition [6]. As other extracted and purified glycosaminoglycans (GAGs), they show high size, mean molecular weight (Mw) 50 kDa and heterogeneous chains [7] too great to be fully characterized as they are. They must be partially/fully depolymerized by enzymatic or chemical methods. Lyase enzymes generally cleave hexosamine–uronic acid bonds and the final products of their exhaustive digestion are disaccharides bearing at the non-reducing end (NRE) a 4,5-unsaturated (∆) uronic acid. DS, known as CS-B, is a variant bearing a number of GlcA units epimerized to L-iduronic acid (L-IdoA), some of them
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