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

Abstract

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.

Combining 1H NMR spectroscopy and multivariate regression techniques to quantitatively determine falsification of porcine heparin with bovine species

“…In the present study we demonstrated that advanced regression techniques, especially PLS and RR are useful tools for discriminating porcine and bovine heparin species and to quantify the contamination level of 2% in positive samples. Our results in combination with some previous studies on detecting and elucidating of heparin impurities and contaminations [20,21,24,26] suggested that NMR spectroscopy is currently the most versatile instrumental approach for authenticity and quality assessment of heparin and heparinoids. to 20% w/w of bovine content.…”

“…2D NMR spectroscopy (DOSY, NOESY, HSQC) as well as measurements at higher temperatures improved the reliability of impurities identification [15]. Later it was shown that NMR outperformed other spectroscopic techniques, such as attenuated total reflection-infrared (ATR-IR) and Raman spectroscopy in heparin characterization [21].…”

“…Recently this method was implemented as a mandatory identity test in European Pharmacopoeia (EP) and US Pharmacopoeia (USP) [18,19]. NMR spectroscopy proved to be suitable to detect adulteration of heparin with chrondroitin sulfate (OSCS) and dermatan sulfate (DS) as well as with other impurities with detection limits smaller than 0.5% [20,21]. 2D NMR spectroscopy (DOSY, NOESY, HSQC) as well as measurements at higher temperatures improved the reliability of impurities identification [15].…”

“…Several papers have demonstrated the utility of chemometric techniques to prove heparin authenticity regarding possible contamination based on NMR profiling[20,21,40,[44][45][46]. For example, principle component analysis (PCA) revealed differences between pure heparin and OSCS-and DC-contaminated samples[20].…”

Low molecular weight heparin biosimilars: how much similarity for how much clinical benefit?

“…En effet, les héparines sont des mélanges de chaînes glycaniques présentant des caractéristiques immunoallergiques (thrombopénie induite par l'héparine) [27] très spécifiques. De plus, la contamination des matières premières par d'autres composés (par exemple chrondroïtine hypersulfatée) ou d'autres glycanes lors de l'extraction de ces molécules est susceptible de déclencher des réac-tions immunitaires potentiellement dramatiques [1]. Enregistrement d'un biosimilaire d'énoxaparine par la FDA En juillet 2010, la FDA a approuvé le premier générique de l'énoxapa-rine (commercialisé sous le nom de Lovenox ® ou de Clexane ® par Sanofi-Aventis).…”