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Following the heparin adulteration crisis, the European Pharmacopoeia (Ph. Eur.) Group of Experts on Biologicals (Group 6) considered a revision of the general chapter 2.7.5. Assay of heparin with regard to the assay of the anticoagulant activity of heparin in order to replace the clotting method with more specific chromogenic methods for anti-IIa and anti-Xa activities. An international collaborative study was carried out in 3 phases under the aegis of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Commission in order to recalibrate Heparin sodium Biological Reference Preparation (BRP) batch 3 for these new assays. Phase 1 confirmed the feasibility of the project, but also indicated that the composition of the buffers affects the assay results, thereby highlighting the importance of using common assay procedures. Phase 2 consisted of a collaborative study involving 15 laboratories to calibrate the anti-IIa and anti-Xa activities of Heparin sodium BRP batch 3. The collaborative study confirmed that Heparin sodium BRP batch 3 is suitable for use as a reference preparation in the proposed chromogenic assays for unfractionated heparin. It also showed that the currently defined acceptance limits (90 % to 111 %) can be maintained in the revised Ph. Eur. texts. Phase 3 of the study collected data on the impact of the new unitage on the release of products marketed in Europe. The data from 5 manufacturers, who each reported results from both the clotting and chromogenic assays for a total of 23 batches, indicated that the replacement of the pharmacopoeial method is unlikely to cause batch release issues. Based on the results of this study, the Ph. Eur. Commission assigned Heparin sodium BRP batch 3 with a potency of 1000 IU/vial for both anti-IIa and anti-Xa activities in the chromogenic assays.

Oversulphated Chondroitin Sulphate (OSCS) and Dermatan Sulphate (DS) in unfractionated heparins can be identified by nuclear magnetic resonance spectrometry (NMR). The limit of detection (LoD) of OSCS is 0.1% relative to the heparin content. This LoD is obtained at a signal-to-noise ratio (S/N) of 2000:1 of the heparin methyl signal. Quantification is best obtained by comparing peak heights of the OSCS and heparin methyl signals. Reproducibility of less than 10% relative standard deviation (RSD) has been obtained. The accuracy of quantification was good.

The proposed European Pharmacopoeial (EP) method for molecular weight determination of low molecular weight heparins (LMWHs) has been shown to have a range which is too narrow to allow for the accurate molecular weight determinations of all LMWHs. We have recently shown that a chemically degraded benzyl ester of unfractionated heparin, ITH-3, is a better calibrator using this methodology. Data is presented here which indicates that this calibrator cannot only be used to determine the molecular weights of LMWHs but also of unfractionated heparins. Therefore, it is now renamed heparin mass calibrator or HMC because of this extension of range. Weight average molecular weight, number average molecular weight, peak molecular weight, and dispersity values were calculated using the HMC calibration and a reference narrow-range 19-calibrator method for various LMWHs and unfractionated heparins. Values for the parameters calculated using the HMC calibration were found not to be significantly different from those of the reference method until the molecular weight exceeded 15.0 kDa. In contrast, the molecular weight profile obtained with the proposed EP method was significantly different from the reference method for samples > 8.0 kDa. The range exhibited by the HMC calibrator should allow it to be used for both LMWHs and unfractionated heparins.