Quantitative Risk Evaluation of Adventitious Agents in Heparin.

Heparin is typically extracted from domestic pigs, which may carry zoonotic adventitious agents. Prion and viral safety cannot be assured by testing the active pharmaceutical ingredient itself; instead for the evaluation of the adventitious agent (i.e., viruses/prions) safety of heparin and heparinoid (e.g., Orgaran or Sulodexide) therapeutics, a risk assessment is required. An approach is presented which provides a quantitative estimation of the worst-case potential residual adventitious agent (i.e., GC/mL or ID 50 ) present in a maximum daily dose of heparin. This estimation is based on the input (determined by prevalence, titer, and amount of starting material to prepare a maximum daily dose) and validated reduction by the manufacturing process, resulting in an estimation of the worst-case potential level of adventitious agent present in a maximum daily dose. The merits of this quantitative, worst-case approach are evaluated. The approach described in this review provides a tool for a quantitative risk evaluation of the viral and prion safety of heparin.

Quantitative 2D 1H, 13C HSQC NMR Spectroscopy for the Determination of Chondroitin Sulfate and Dermatan Sulfate Content in Danaparoid Sodium.

OBJECTIVE: Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph. Eur. The monograph includes the CS and DS limit contents and describes the method for their quantification through selective enzymatic degradations. MATERIALS AND METHODS: In this study, a quantitative two-dimensional nuclear magnetic resonance (NMR) method is proposed as a new method suitable for CS and DS quantification. Statistical comparison of the results provided by the analysis of a series of danaparoid samples with both NMR and enzymatic methods highlights a small systematic difference, likely derived from lyase-resistant sequences bearing oxidized terminals. Some modified structures, whose survival to the enzymatic action was confirmed by mass spectrometry, can be detected and quantified by NMR. CONCLUSION AND RESULTS: The proposed NMR method can serve for the determination of DS and CS contents, is an easy-to-apply method with no dependence from enzymes and standards, and provides extensive structural information on the overall glycosaminoglycans mixture.

Selective Binding of Heparin/Heparan Sulfate Oligosaccharides to Factor H and Factor H-Related Proteins: Therapeutic Potential for C3 Glomerulopathies.

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.

The conformation of the idopyranose ring revisited: How subtle O-substituent induced changes can be deduced from vicinal 1H-NMR coupling constants.

The idopyranose ring plays a pivotal role in the conformational, dynamical, and intermolecular binding aspects of glycosaminoglycans like heparin and dermatan sulfate and it was early on assigned a role in the Sugar Code governing biological recognition processes. There is consensus that next to the two canonical 1C4 and 4C1 chair conformations, the conformational space accessible to the idopyranose ring entails a 2SO skew-boat conformation, but the equilibrium between these three ring puckers has evaded satisfactory quantification. In this study a meta-analysis of X-ray solid-state data and vicinal NMR coupling constants is presented, based on the Truncated Fourier Puckering (TFP) formalism and the generalized Karplus (CAGPLUS) equation. This approach yields a model-free, granular and consistent reckoning of 159 idopyranose solution puckering equilibria studied by NMR and allows us to reproduce the involved 636 NMR vicinal couplings with an overall residual RMS(Jobs-Jcalc) of 0.184 Hz. Our analyses show that for all ring systems examined, the idopyranosyl chair conformations take up the same ring pucker irrespective of the ring substituent pattern or a vast variety in experimental conditions. Instead, it is the (skew-)boat conformation that adapts to the substitution pattern of the idopyranose ring or a specific sulfation pattern of neighboring saccharides. All idopyranose rings are involved in conformational equilibria that subsume the aforementioned conformers which turn out to differ only a few kJ/mole in conformational energy. Thus, the plasticity and flexibility of idopyranose remains intact under practically all circumstances and, as the glycosidic linkages in heparin are considered to be relatively stiff, the iduronic moiety functions as the linchpin of heparin flexibility thereby being rather a "space(r)" than a "letter" in the alleged Sugar Code alphabet.

Extended Physicochemical Characterization of the Synthetic Anticoagulant Pentasaccharide Fondaparinux Sodium by Quantitative NMR and Single Crystal X-ray Analysis.

Fondaparinux sodium is a synthetic pentasaccharide representing the high affinity antithrombin III binding site in heparin. It is the active pharmaceutical ingredient of the anticoagulant drug Arixtra®. The single crystal X-ray structure of Fondaparinux sodium is reported, unequivocally confirming both structure and absolute configuration. The iduronic acid adopts a somewhat distorted chair conformation. Due to the presence of many sulfur atoms in the highly sulfated pentasaccharide, anomalous dispersion could be applied to determine the absolute configuration. A comparison with the conformation of Fondaparinux in solution, as well as complexed with proteins is presented. The content of the solution reference standard was determined by quantitative NMR using an internal standard both in 1999 and in 2016. A comparison of the results allows the conclusion that this method shows remarkable precision over time, instrumentation and analysts.

From Farm to Pharma: An Overview of Industrial Heparin Manufacturing Methods.

The purification of heparin from offal is an old industrial process for which commercial recipes date back to 1922. Although chemical, chemoenzymatic, and biotechnological alternatives for this production method have been published in the academic literature, animal-tissue is still the sole source for commercial heparin production in industry. Heparin purification methods are closely guarded industrial secrets which are not available to the general (scientific) public. However by reviewing the academic and patent literature, we aim to provide a comprehensive overview of the general methods used in industry for the extraction of heparin from animal tissue.

The use of 13C labeling to enhance the sensitivity of 13C solid-state CPMAS NMR to study polymorphism in low dose solid formulations.

(13)C labeling was used to enhance the sensitivity of (13)C solid-state NMR to study the effect of tabletting on the polymorphism of a steroidal drug. The steroidal drug Org OD 14 was (13)C labeled and formulated into tablets containing only 0.5-2.5% active ingredient. The tablets were subsequently studied by solid-state (13)C CPMAS NMR. The crystalline form present in tablets could readily be analyzed in tablets. No change in crystalline form was observed as a result of formulation or in subsequent stability studies. Solid-state NMR in combination with (13)C labeling can, in suitable cases, be used as a strategy to study the effect of formulation on the polymorphism of low dose drugs.