SAX-HPLC and HSQC NMR Spectroscopy: Orthogonal Methods for Characterizing Heparin Batches Composition.

Heparin is a complex mixture of heterogeneous sulfated polysaccharidic chains. Its physico-chemical characterization is based on the contribution of several methods, but advantages of the use of complementary techniques have not been fully investigated yet. Strong-Anion-Exchange HPLC after enzymatic digestion and quantitative bidimensional 1H-13C NMR (HSQC) are the most used methods for the determination of heparin structure, providing the composition of its building blocks. The SAX-HPLC method is based on a complete enzymatic digestion of the sample with a mixture of heparinases I, II and III, followed by the separation of the resulting di- and oligo-saccharides by liquid chromatography. The NMR-HSQC analysis is performed on the intact sample and provides the percentage of mono- and di-saccharides by integration of diagnostic peaks. Since, for both methods, accuracy cannot be proved with the standard procedures, it is interesting to compare these techniques, highlighting their capabilities and drawbacks. In the present work, more than 30 batches of porcine mucosa heparin, from 8 manufacturers, have been analyzed with the two methods, and the corresponding results are discussed, based on similarities and differences of the outcomes. The critical comparison of both common and complementary information from the two methods can be used to identify which structural features are best evaluated by each method, and to verify from the concordance of the results the accuracy of the two methods, providing a powerful tool for the regular characterization of single, commercial preparations of Heparin.

Capillary electrophoresis method for speciation of iron (II) and iron (III) in pharmaceuticals by dual precapillary complexation.

Pharmaceutical iron sucrose is an iron (III) replacement for the treatment of iron deficiency anemia in patients with chronic kidney disease. The drug product (injection) is a colloidal solution of ferric hydroxide in complex with sucrose, containing 20 mg/mL elemental iron; according to United States pharmacopoeia (USP), the limit of iron (II) is 0.4% w/v. A selective CE method for the simultaneous determination of iron (III) and its potential impurity iron (II), was developed by applying a dual precapillary complexation. In particular, 1,10-phenanthroline and 1,2-diaminocyclohexanetetraacetic acid were used for complexation of iron (II) and iron (III), respectively. Sample preparation was optimized to achieve mineralization of pharmaceuticals using HCl 6 M, by avoiding perturbation of the oxidation status of both iron species. Simple CZE conditions, involving a 60 mM (pH 9.3) tetraborate buffer at the constant voltage of 25 KV and 25°C, allowed fast separation of iron (II) and iron (III) complexes that were detected at 265 nm. Sensitivity for iron (II) determination was found to be 4.80 µM (LOQ) corresponding to 0.15% w/w with respect to the total iron test level. The method was validated by following International Conference on Harmonization guidelines for specificity, linearity, precision, accuracy, and robustness and it was applied to real pharmaceutical samples. The obtained results suggested that the method can be a useful alternative to the official USP and British pharmacopoeia polarographic method.

Affinity capillary electrophoresis in binding study of antithrombin to heparin from different sources.

Heparin, a highly sulfated polydispersed glycosaminoglycan (GAG), is the most widespread clinical anticoagulant; it binds antithrombin III (AT), a member of serine proteinases superfamily, accelerating its antagonist effect on blood coagulation. The binding interaction with AT is an important aspect in characterization of physicochemical properties of GAGs. With the aim at profiling several clinical and experimental heparin batches from different sources (porcine, bovine and ovine mucosa), a quantitative investigation of the binding heparin-AT, was undertaken by means of Affinity Capillary Electrophoresis (ACE). In dynamic-equilibrium ACE, the electrophoretic mobility of the receptor (AT), analyzed in a BGE containing the ligand (the considered GAG), is correlated to ligand concentration and binding constant. In particular, a 20mM sodium phosphate, pH 7.4 buffer (the BGE) was chosen as the neat medium and the experiments were carried out in a highly hydrophilic poly(vinyl alcohol) coated capillary (effective length 8.5 cm). The applied sample, consisting in the receptor AT (0.30 µM) and phenylacetic acid (PAA; 10.0 µM) used as a reference compound, was electrophoresed at each of the studied concentration levels of the ligand (heparin samples, 0.30-10.0 × 10(-7)M; heparan sulfate, 0.35-8.0 × 10(-5)M) supplemented to the BGE. The migration time ratio of PAA to AT was assumed as the chemical response to be correlated to the ligand concentration and the binding constant estimation was based on the application of a nonlinear regression method (rectangular hyperbola). Under these conditions, a number of heparin samples were analyzed and their binding constants (Kd) were found within 14.2 and 56.1 nM (SD ≤ ± 2.0; n=3; coefficient of determination r(2) ≥ 0.96). The good correlation of Kd values to the in-vitro activity (anti-factor Xa and anti-factor IIa), confirmed that the affinity for the target AT is an important feature of heparin samples and could be included among their physico-chemical characteristics.

Determination of dermatan sulfate and chondroitin sulfate as related substances in heparin by capillary electrophoresis.

Capillary electrophoresis (CE) was applied to the quantitation of dermatan sulfate (DS) and chondroitin sulfate (CS) as related substances in sodium heparin. The method is based on the selective digestion of either CS and DS contained in the main drug heparin, by using chondroitinase ABC (specific for both DS and CS) and chondroitinase AC (specific for only CS). The unsaturated disaccharides released after exhaustive digestion, can be separated by CE using a 110mM phosphate buffer, pH 3.5 as the background electrolyte in a fused silica capillary (64.5cmx50mum i.d.) at 40 degrees C and -30kV. Since the level of each disaccharide released upon enzymatic digestion corresponds to its content in the native glycosaminoglycan, the amount of CS and DS was determined by proportion with the released disaccharides. In particular, DeltaUA-->GalNAc-4S Na(2) and DeltaUA-->GalNAc-6S Na(2) were selected for quantitation of CS and DS because of their significant response and short migration time (less than 7min).The method was validated for linearity, accuracy, precision and it showed to be able in detecting selectively, DS and CS at impurity level (LOD 0.01%, w/w). The proposed CE approach was finally applied to real samples. The results obtained were found in excellent correlation with those achieved by the analysis of the same samples using the official USP method based on high performance anion exchange chromatography (HPAEC) with pulsed amperometric detector.

Low-molecular-weight heparin from Cu2+ and Fe2+ Fenton type depolymerisation processes.

Hydrogen peroxide (H2O2) and Cu(OAc)2 or FeSO4 (Fenton type reagents) perform heparin (Hep) depolymerisation to low-molecular-weight heparin (LMWH) following a radical chain mechanism. Hydroxyl (OH) radicals which are initially generated from H2O2 reduction by transition metal ions abstract hydrogen atoms on the heparin chain providing carbon centred radicals whose decay leads to the depolymerisation process. The main depolymerisation mechanism involves Hep radical intermediates that cleave the glycosidic linkage at unsulphated uronic acids followed by a 6-O-nonsulphated glucosamine, thus largely preserving the pentasaccharide sequence responsible for the binding to antithrombin III (AT). Both the transition metal ions influence the overall efficiency of the radical chain processes: Fe2+ acting as a catalyst, while Cu2+ acts as a reagent. LMWHs, especially those afforded by Cu2+, are somewhat unstable to the usual basic workup. However, this lack of stability can be eliminated by a previous NaBH4 reduction. Furthermore, with Cu2+, the process is much more reproducible than with Fe2+. Therefore, for the process of Fenton type depolymerisation of heparin, the use of Cu(OAc)2 is clearly preferable to the more "classical" FeSO4. The resulting activities and characteristics of these LMWHs are peculiar to these oxidative radical processes. In addition, LMWH provided by H2O2/Cu(OAc)2 in optimised conditions was found to posses anti-Xa and anti-IIa activities comparable to those of LMWHs currently in clinical use.

Heparins: process-related physico-chemical and compositional characteristics, fingerprints and impurities.

During the past 25 years, heparin extraction and purification processes have changed. The results of these changes are reflected by the continuous increase in potency of the International Standard for heparin. This increase is due not only to a higher purity, but also to a number of changes in the physico-chemical characteristics of heparin. For long time, all these changes have been disregarded as non-critical by regulatory authorities. Heparin marketing authorisation was reviewed only two years ago and Pharmacopoeia monographs were reviewed just for the addition of new tests, mainly aimed at tackling the oversulfated chondroitin sulfate (OSCS) crisis. Currently, heparin monographs are again under revision. Such changes, different for each manufacturer, have caused a further increase in the heterogeneity of individual batches of heparin. This review aims at showing that chemical, physical and biological characteristics of heparin (such as disaccharide composition, amount of low sulfated and high sulfated sequences, molecular weight profiles [MW], activities, structural artifacts, fingerprints and glycosaminoglycans impurities) are all process-dependent and may significantly vary when different processes are used to minimise the content of dermatan sulfate. The wide heterogeneity of the physico-chemical characteristics of currently marketed heparin and the lack of suitable and shareable reference standards for the identification/quantification of process-related impurities caused, and are still causing, heated debates among scientific institutions, companies and authorities.

Structural modification induced in heparin by a Fenton-type depolymerization process.

A low molecular weight heparin (LMWH) obtained by a depolymerization process induced by a Fenton-type reagent was characterized in depth by nuclear magnetic resonance (NMR) spectroscopy. The depolymerization involves the cleavage of glycosidic bonds, leading to natural terminal reducing end residues, mainly represented by N-sulfated glucosamine (A (NS)). Natural uronic acids, especially the 2- O-sulfate iduronic acid (I (2S)), are also present as reducing residues. A peculiar reaction results, such as the disappearance of the nonsulfated iduronic acid residues when followed by 6-O-nonsulfated glucosamine, and the decrease of the glucuronic acid when followed by the N-acetylglucosamine, were observed. Iduronic acid residues, followed by 6- O-sulfate glucosamine (A (Nx,6S)), and the glucuronic acid residues, followed by A (NS) residues, were not modified. A few minor internal chain modifications occur, possibly arising from oxidative breaking of the bond between C2-C3 of glucosamine and uronic acids, suggested by evidence of formation of new -COR groups. Finally, no change was observed in the content of the N-sulfated, 6-O-sulfated glucosamine bearing an extra sulfate on 3-O, which is considered the marker of the active site for antithrombin. With respect to the original heparin, this LMWH is characterized by a lower number of nonsulfated uronic acid residues, and as a consequence, by a lower degree of structural heterogeneity than LMWHs prepared with other procedures.

Variability of heparins and heterogeneity of low molecular weight heparins.

Chemical and physical characteristics, building blocks, constitutive disaccharides, sulfation degree, and biological activities of heparins (UFHs) and of low molecular weight heparins (LMWHs) obtained by different depolymerization processes are examined comparatively in terms of structure characteristics, content of 1,6-anhydro rings, and other fingerprints. The heterogeneity of different LMWHs depends on different manufacturing processes and on particular specifications of pharmacopoeias. The reported examples prove that the variability among samples of LMWHs manufactured by the same process is quite limited. Most of the variability is derived from the parent UFH. In contrast, fingerprint groups and residues are specific to the depolymerization process and their extent can be roughly controlled through the process parameters.

Characterization of di- and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1,6-anhydro-beta-D-glucosamine or N-sulfated 1,6-anhydro-beta-D-mannosamine residues.

Modified heparin disaccharides were obtained by the alkaline treatment of a solution containing the disulfated heparin disaccharide DeltaHexA-alpha-(1-->4)-D-GlcNSO(3),6SO(3). Their structures were characterized by one- and two-dimensional NMR spectroscopy: DeltaHexA-alpha-(1-->4)-1,6-anhydro-GlcNSO(3), DeltaHexA-alpha-(1-->4)-1,6-anhydro-ManNSO(3) and DeltaHexA-alpha-(1-->4)-ManNSO(3),6OSO(3). NMR spectroscopy, in combination with HPLC, provided the composition of the mixture. Characteristic NMR signals of the disaccharides were identified, even at low levels, in a high field of (1)H-(13)C correlation NMR spectra (HSQC) of a low molecular weight heparin (LMWH) obtained by beta-elimination (alkaline hydrolysis) of heparin benzyl ester, providing a more complete structural profile of this class of compounds.

Free radical generation during chemical depolymerization of heparin.

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.

Sepharose-bound, highly sulfated glycosaminoglycans can capture HIV-1 from culture medium.

In the search for new strategies against HIV-1 and on the basis of a number of previous studies reporting on the capacity of certain polyanionic compounds to influence the replication of HIV-1, we prepared a few chemically oversulfated dermatan and chondroitin sulfates. Four of these compounds and two samples of heparin were bound to activated Sepharose through either their carboxylic groups, or their aldehydic groups, or their deacetylated primary amino groups. Some of these so-derivatised resins, packed into columns, proved able to remove HIV-1 IIIB, a laboratory adapted strain, and one clinical primary isolate from an AIDS patient, from infected cell culture medium. The resins bind the virus very tightly and could be useful for capturing the virus from infected fluids.

Susceptibility to highly sulphated glycosaminoglycans of human immunodeficiency virus type 1 replication in peripheral blood lymphocytes and monocyte-derived macrophages cell cultures.

In the search for new drugs against human immunodeficiency virus type 1 (HIV-1), the replication of III(B) and BaL strains, and of seven primary isolates from AIDS patients, cultured both in peripheral blood lymphocytes (PBLs) and in monocyte-derived macrophages (MACs), was investigated in the presence of two dermatan sulphate and heparin at 10 microg/ml. The three polysaccharides effectively inhibited the replication of III(B) in PBLs and of BaL in MACs, while producing either a slight inhibition or an unexpected large increase in the replication of the seven primary isolates, especially in MAC cultures. In one case, stimulation was found in PBLs and, at lower doses, also with BaL in MACs. Co-receptor use, adaptation to C8166 T cell line, partial sequence of the gp120 V3 loop, variation in positive charge distribution and number of potential glycosylation sites along the V3 loop were assessed for each strain. No explanation could be found for the different susceptibility of the viruses to the polysaccharides. Their presence probably brings about both inhibitory and stimulatory effects, the final outcome depending on the virus, cells and polysaccharide.