Simple and rapid quality control of sulfated glycans by a fluorescence sensor assay--exemplarily developed for the sulfated polysaccharides from red algae Delesseria sanguinea.

Sulfated polysaccharides (SP) from algae are of great interest due to their manifold biological activities. Obstacles to commercial (especially medical) application include considerable variability and complex chemical composition making the analysis and the quality control challenging. The aim of this study was to evaluate a simple microplate assay for screening the quality of SP. It is based on the fluorescence intensity (FI) increase of the sensor molecule Polymer-H by SP and was originally developed for direct quantification of SP. Exemplarily, 65 SP batches isolated from the red alga Delesseria sanguinea (D.s.-SP) and several other algae polysaccharides were investigated. Their FI increase in the Polymer-H assay was compared with other analytical parameters. By testing just one concentration of a D.s.-SP sample, quality deviations from the reference D.s.-SP and thus both batch-to-batch variability and stability can be detected. Further, structurally distinct SP showed to differ in their concentration-dependent FI profiles. By using corresponding reference compounds, the Polymer-H assay is therefore applicable as identification assay with high negative predictability. In conclusion, the Polymer-H assay showed to represent not only a simple method for quantification, but also for characterization identification and differentiation of SP of marine origin.

Testing of potential glycan-based heparanase inhibitors in a fluorescence activity assay using either bacterial heparinase II or human heparanase.

Heparanase, an endo-ß-glucuronidase cleaving heparan sulfate (HS) chains at cell surfaces and in the extracellular matrix (ECM), is involved in angiogenesis, tumor progression and metastasis as well as in inflammation and kidney dysfunction. Therefore, heparanase is considered a promising therapeutic target and diagnostic marker. Recently, we have developed a simple, rapid, fully automatable fluorimetric activity assay using the synthetic sulfated pentasaccharide fondaparinux as substrate and bacterial heparinase II (HEP-II) instead of human heparanase (hHEP). The aim of this study was to evaluate this assay for inhibitor testing as well as to check whether the assay principle is applicable to measure the activity and inhibition, respectively, of the actual target enzyme hHEP. Besides the known hHEP inhibitor suramin and the antiinflammatory and antimetastatic PS3, two series of ß-1,3-glucan sulfates differing in their chain length and degree of sulfation, further semisynthetic sulfated glycans, and two sulfated polysaccharides isolated from algae were included to examine structure-activity relationships. The inhibitory activity of sulfated glycans showed to be greatly dependent on both their degree of sulfation and their basic glycan structure, but independent of their molecular size. The ß-1,3-glucan sulfates were superior to suramin as well as to the other glycans with similar degree of sulfations. The most active inhibitor was found to be the ß-1,3-glucan sulfate PS3 (IC50=0.017 µM). By using hHEP instead of HEP-II comparable results were obtained. With an IC50 being about 160 times lower than that of suramin, PS3 exhibited again the strongest inhibitory effects. Inhibition of hHEP may therefore contribute to the potent antiinflammatory and antimetastatic activities of PS3 in vivo. In conclusion, the fluorimetric hHEP activity assay proved to be a simple, fully automatable tool for testing potential inhibitors. In case of HS mimetic inhibitors, the assay variant with HEP-II may provide a fast and inexpensive option for initial screening purposes.

Development of both colorimetric and fluorescence heparinase activity assays using fondaparinux as substrate.

Because tumors and other diseases are characterized by increased heparanase levels, human heparanase is a promising drug target and diagnostic marker. Therefore, methods are needed to determine heparanase activity and to examine potential inhibitors. Because of substrate comparability, we used the bacterial enzyme heparinase II (heparinase) for the assay development. Usually the substrate of heparanase assays is heparan sulfate, which has several disadvantages. Because of that, we used fondaparinux, which is being cleaved by both heparanase and heparinase. Two concepts to detect its degradation were examined: measurement of anti-factor Xa activity of fondaparinux and its direct quantification with the fluorescent sensor polymer-H. Using fondaparinux as substrate, the anti-factor Xa assay was shsown to be appropriate to determine heparinase activity. The detection with polymer-H was easier and even faster to perform. Linearity was given with fondaparinux as well as heparan sulfate, and heparin as substrates, but fondaparinux turned out to be most suitable. By modifications (incubation time, fondaparinux concentration, and polymer-H concentration), the limit of quantification and the linear range can be adapted to the respective requirements. In conclusion, a simple, accurate, and robust heparinase assay was developed. It is suitable for heparinase quality control and testing heparinase inhibitors and could be adapted to heparanase.

Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples.

The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides (1)H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had <180 IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results.

Simple fluorescence assay for quantification of OSCS in heparin.

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ ((em)) 330 nm, λ ((ex)) 510 nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with (1)H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect.

Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins.

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 option - indirect 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.

Development and evaluation of a fluorescence microplate assay for quantification of heparins and other sulfated carbohydrates.

Due to their complex composition, quantification of heparins is difficult. On the one hand there are many biological tests, which only indirectly detect effects of the antithrombin-binding material. On the other hand direct quantitative methods are available but they are often insensitive, challenging, time-consuming or expensive. The aim of this study was to develop a sensitive, rapid, simple as well as inexpensive direct quantification assay suitable for routine analysis. Based on Polymer-H, a novel heparin complexing, fluorescent labeled synthetic polymer (lambda((ex)) 320nm, lambda((em)) 510nm), a microplate assay was developed and optimized. The specificity of the assay was evaluated by structure-assay response relationships studies using structurally defined glucan sulfates, heparins, and other natural and synthetic sulfated carbohydrates. The fluorescence intensity of Polymer-H (7.5microg/ml) showed to be concentration-dependently amplified by heparins as well as by other sulfated carbohydrates. The best sensitivity, accuracy and linearity were observed in a range from 0.63 to 5.0microg/ml heparins. No differences in the fluorescence between various heparins were observed, so that only one calibration curve is needed. In addition, all types of carbohydrates with a degree of sulfation (DS)> approximately 1.2 and a M(r)>3000 can be quantified as well. By own calibration curves also other sulfated carbohydrates like fondaparinux or other glycosaminoglycans (DS>0.4) can be determined.