Molecular and Functional Heterogeneity in Contaminants Isolated from Recalled Heparin. Impact on Anticoagulation and Potential Adverse Reactions

Abstract

The main contaminant in the recalled batches of unfractionated heparin (UFH) is reported to be oversulfated chondroitin sulfate (OSCS). No data on the composition and biologic properties of this reported OSCS is available. Moreover, it has been assumed that different batches of preparations contained similar forms of OSCS. Since this contaminant has been purposefully added to UFH and has been found in several batches from various suppliers and is also found in some of the low molecular weight heparins (LMWHs), it was hypothesized that some type of OSCS was used to supplement UFH preparations. To test this hypothesis four batches of contaminated UFH and two batches of LMWH were investigated. Four batches of UFH of the recalled products from the US suppliers and two batches of a LMWH from the European community were compared. The molecular weight profile studies were carried out using high performance liquid chromatographic methods. Each of these products were subjected to heparinase-1 digestion to determine the non-digestable components. The anticoagulant activities of each of these products were measured using the whole blood activated clotting time and thromboelastographic analysis. The effect of each product was also studied on thrombin generation (Fibrinopeptide A, thrombin antithrombin complex, and prothrombin F1.2) and contact activation. The anticoagulant assays (PT, APTT, TT, Heptest, ecarin clotting time), antiprotease profile (anti-Xa and anti-IIa) and thrombin generation inhibitory studies were carried out in the normal human plasma. Protamine and platelet factor 4 neutralization were also carried out in plasma. Non-heparin contaminants were also isolated from each of the heparins by digestion of heparin followed by alcohol precipitation and ion exchange chromatographic methods. The isolated components were further purified by physicochemical methods. Each of these components were profiled for the molecular, structural and biologic activity. In addition each of the contaminants were further characterized in terms of their interactions with SERPINS (AT III and HCII), platelet factor 4 and analyzed for anticoagulant activity in whole blood and citrated plasma systems. The four contaminated UFHs (H1-H4) did not exhibit any major differences in the molecular weight profile (14.8–15.6 KDa). The USP potency of these products were also similar (158–170 U/mg). The anticoagulant actions were comparable in the different whole blood and global assays. However, the anti-Xa and anti-IIa ratios were found to exhibit some variations (0.93–1.24). Each product showed differences in the heparinase resistant component (14–30%). H1 and H3 products also contained significant amounts of dermatan sulfate. Each of the contaminants exhibited different neutralization profiles with PF4 and protamine sulfate. The LMWH products were comparable in all of the studies including the molecular weight profile and biologic actions, however, they showed differences in the heparinase-1 digestion profiles. Moreover, the molecular weight of the contaminant obtained from the LMWH was lower (12.8 vs. 14.1–16.8 KDa). The contaminants also exhibited differences in thrombin generation markers. The USP potency of the heparin contaminants varied from 28–46 U/mg whereas the contaminant from the LMWH exhibited a potency of 38–46 USP U/mg. These studies suggest that the contaminants isolated from recalled batches of heparin are heterogenous in nature and may originate from multiple sources. Moreover, the contaminants obtained form LMWHs may exhibit additional structural and biologic differences. Therefore, the wide variations observed in the adverse reactions with recalled heparins may be due to compositional variations in the contaminants.