Impact of autoclave sterilization on the activity and structure of formulated heparin (original) (raw)
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Chemical Microdetermination of Heparin in Plasma
Analytical Biochemistry, 1997
introduced, it was noted that an individual calibration A new, simple, and highly sensitive method for the curve was required for each heparin preparation (4). determination of heparin has been established. Hepa-This point has often been ignored resulting in considerrin was first converted into unsaturated disaccharides able variation in coagulation response to different hepthrough the action of heparin lyases I, II, and III. A arin preparations. Differences are often observed bemajor trisulfated unsaturated disaccharide product tween the amount of heparin measured by coagulation results, consistent with structural analysis of a numtests and the actual in vivo drug concentration (5). ber of pharmaceutical heparins using one-and two-While chemical measurements of heparin in plasma dimensional 1 H NMR spectroscopy. This disaccharide have been proposed, they generally lack sufficient senwas analyzed by HPLC using fluorometric postcolumn sitivity to be clinically useful (6, 7). A sensitive method derivatization. The correlation between the amount of that measures the amount of chemical heparin in biothis trisulfated unsaturated disaccharide and anticological samples would be of great importance in reguagulant activity of heparin as measured by anti-IIa lating its dose and preventing side effects. This paper was determined. The analysis of these pharmaceutical describes such a simple and sensitive procedure for the heparins showed a linear correlation between both determination of heparin in plasma. Heparin is enzy-HPLC and bioassay. This HPLC method was then apmatically depolymerized and its major trisulfated unplied to a pharmacokinetic study of heparin intravesaturated disaccharide is analyzed by HPLC using nously administered to rabbits. ᭧ 1997 Academic Press postcolumn derivatization. This method has been applied to the pharmacokinetic study of heparin intravenously administered to rabbits. Heparin is a polydisperse, highly sulfated, linear MATERIALS AND METHODS polysaccharide composed of repeating 1 r 4 linked uronic acid and glucosamine residues (1). When pharma-Six different heparin samples, sodium salts, from ceutical heparin is subjected to consecutive fractionporcine and bovine intestinal mucosal and bovine lung ation by chromatography or electrophoresis, it is found heparin were obtained from Sigma Chemical Co. (St. to consist of many different, closely related, sulfated Louis, MO), Nacalai Chemical Co. (Tokyo, Japan), polysaccharides of which only a fraction have anticoag-Fluka Chemika-Biochemika Japan Co. (Tokyo, Japan), ulant activity (2). Heparin also has another level of and in large quantities from Celsus Laboratories (Cinstructural complexity, associated with its primary cinnati, OH). Low-molecular-weight heparin (average structure or sequence (3). Thus, the failure to commolecular weight, 6000) prepared from bovine intestipletely understand heparin's structure is not the result nal mucosa was purchased from Sigma. Heparin disacof a lack of effort but rather is due to it's extremely charide standards were from Seikagaku Co. (Tokyo, complex structure (3). Japan), Sigma, Dextra Laboratories (Reading, UK), or The presence of heparin in biological fluids and drugs Grampian Enzymes (Aberdeen, Scotland). Heparin lyis usually estimated by bioassays such as coagulation ase I (EC 4.2.2.7), heparin lyase II (No EC assigned), tests (4). When these coagulation tests were originally and heparin lyase III (EC 4.2.2.8) from Flavobacterium heparinum were from Seikagaku, Grampian Enzymes, or Sigma.
Analytical Biochemistry, 2002
Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein-heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.
Analytical biochemistry, 2014
The thermal instability of the anticoagulant heparin is associated, in part, with the solvolytic loss of N-sulfo groups. This study describes a new method to assess the increased content of unsubstituted amino groups present in thermally stressed and autoclave-sterilized heparin formulations. N-Acetylation of heparin samples with acetic anhydride-d6 is followed by exhaustive heparinase treatment and disaccharide analysis by hydrophilic interaction chromatography mass spectrometry (HILIC-MS). The introduction of a stable isotopic label provides a sensitive probe for the detection and localization of the lost N-sulfo groups, potentially providing valuable insights into the degradation mechanism and the reasons for anticoagulant potency loss.
PLoS ONE, 2011
The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities.
Method development for studying the interactions between antithrombin and heparin
2008
Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilis...
Analytical and Bioanalytical Chemistry, 2010
There are several methods for sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. Although contamination with OSCS is unlikely to be repeated, use of other compounds to counterfeit heparin must be considered. We have previously developed a two-step fluorescence microplate assay (two-step FI assay) for detection of OSCS. First, the heparin sample is incubated with heparinase I, then its increasing effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H is measured (PolyH assay). The high sensitivity of the assay is shown to be based on heparinase I inhibition by OSCS. The objective of this study was to evaluate another assay optionindirect quantification of OSCS after heparinase I incubation by means of the anti-Factor Xa (aXa) activity of the remaining undegraded heparin (two-step aXa assay). We also examined, whether other heparin mimetics (HepM), direct Factor Xa inhibitors (DXI), and protein impurities are detectable by use of these assays. Heparin was spiked with different amounts of HepM including OSCS, pentosan polysulfate, dextran sulfate, curdlan sulfate, the natural contaminant dermatan sulfate, the DXI rivaroxaban, and BSA as a protein. These samples were compared with pure heparin in the two-step FI assay, the two-step aXa assay, and in the PolyH assay and the aXa assay without heparinase I incubation. Both two-step assays sensitively measured contamination with all the HepM (LOD≤0.5%, LOQ ≤0.7%). The two-step aXa assay also detected rivaroxaban (LOD 0.3%, LOQ 0.4%), whereas the two-step FI assay was shown to be suited to determination of protein impurities (LOD 0.11%, LOQ 0.13%). Use of two different heparinase I inactivation procedures enabled clear differentiation between protein, HepM, and both contaminants. Finally, with the aXa assay the heparin potency can be determined in the same assay run, whereas the FI increase in the PolyH assay was shown to be useful for identification. In conclusion, both the two-step FI assay and the two-step aXa assay are sensitive, rapid, and simple tests for the detection of counterfeit heparin. Comprehensive information about heparin quality can be obtained by their combined use and the parallel measurement of non-incubated heparin samples.