Validation of the Bioequivalence of USP Potency Adjusted Porcine, Ovine, and Bovine Heparins

ABSTRACT

Introduction: Currently, there is a shortage of porcine heparin due to several factors such as limited availability of porcine mucosa, supply chain issues, and increased usage due to COVID-19. This has warranted the development of heparin from alternate sources such as bovine and ovine mucosa which is abundantly available for this purpose. On a mass basis, commercially available porcine heparins exhibit a similar potency (200 units/mg) to their ovine counterpart (190 units/mg) and a higher potency in contrast to their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in various laboratory assays in comparison to bovine heparin and similar effects in comparison to ovine heparins. Since heparin is standardized in biologic units and cross referenced against USP or EP Standard, it is hypothesized that potency equated porcine, ovine, and bovine heparin will exhibit similar biologic activities in laboratory assays carried out in the in vitro setting. The purpose of this study is to compare the biologic properties of the porcine, ovine, and bovine heparin at USP potency equated levels in standardized laboratory assays. Materials and Methods: Active pharmaceutical ingredients (API) of porcine mucosal heparin (200 units/mg) of U.S. origin was commercially obtained from Medefil Inc. (Glendale Heights, IL). Ovine heparin was obtained from Ronnsi Pharmaceutical (Jiangsu, China). Bovine heparin (140 units/mg) was obtained from Kin Master Pharmaceuticals (Posso Fundo, Brazil). All heparins were diluted at a concentration of 100 units/mL in saline. The anticoagulant effect of all heparins were evaluated using the whole blood clotting assays such as the ACT and thromboelastographic methods. Heparins were diluted in citrated human plasma yielding a final concentration range of 0-1 unit/mL. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were measured. Thrombin generation inhibition assay was carried out using a kinetic assay (CAT system, Diagnositca Stago, Paris, France). Protamine and heparinase neutralization profiles of these agents were also investigated in the plasma-based systems. These assays were then repeated at gravimetric dosages at final concentrations of 0-10 ug/mL. The results collected from these trials were then mathematically converted to units and compared to the results collected from the potency adjusted trials. All results were tabulated and compared, and applicable statistical methods were applied. Results: The USP potency adjusted heparin exhibited comparable anticoagulant effects in both the ACT and TEG assays. At equigravimetric levels porcine and ovine heparins produced comparable anticoagulant effects and bovine heparin produced weaker anticoagulant effect in both assays. In the citrated plasma supplementation studies, all drugs produced similar anticoagulant effects at potency adjusted dosages. In the chromogenic anti-Xa and anti-IIa assays, the behaviors of the agents were also comparable. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, the porcine, ovine, and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine, ovine, and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. At gravimetric concentrations, bovine heparins exhibited lower potencies than both the porcine and ovine heparins, which produced similar results. Summary and Conclusion: These results show that at potency adjusted concentrations, the porcine, ovine, and bovine heparin exhibit comparable biochemical and anticoagulant responses in the plasma-based systems. Therefore, the hypothesis that potency equated porcine, ovine, and bovine heparins exhibit comparable biochemical and anticoagulant activities is validated. Thus, the proposed ap roach to standardize heparins against a common standard in a biologic assay such as the USP method is valid. Furthermore, these results warrant regulatory considerations to fast track the review process for the re-introduction of bovine heparin and approval of bovine heparin as a biosimilar anticoagulant to porcine heparin. Disclosures No relevant conflicts of interest to declare.