Abstract
Introduction: Heparin prevents blood clots from forming in patients undergoing heart surgeries, dialysis, multiple other procedures, and for medical treatment of thrombosis such as associated with cancer. Currently, all heparin products in the U.S. are derived from the intestinal mucosa of pigs. Seventy-five percent of the crude porcine heparin used to make the active pharmaceutical ingredient (API) comes from outside the U.S., with a majority originating from China. Reintroduction of bovine heparin into the U.S. market would expand sources for this critical drug, thus addressing concerns about potential shortages and product adulteration. This study was undertaken to determine the bioequivalence of bovine and porcine heparins using assays relevant to the clinical setting. The assays used in this study were chosen because they overcome limitations of the conventional APTT for heparin monitoring. Selected TEG and ACT assays use whole blood which better simulates physiologic conditions and assesses the full in vitro anticoagulation potential of heparin; these assays are also routinely used clinically for heparin monitoring. The thrombin generation assay is state-of-the-art for hemostatic clinical lab testing, and it provides a more sensitive endpoint of the final generation of thrombin once coagulation is activated. Heparins were studied at concentrations used clinically and tested by assays sensitive to these concentrations.
Methods: Bovine heparin API (BH; 7 lots) and US clinical grade porcine heparin (PH: 3 lots) were tested in parallel using recalcified whole blood thromboelastography (TEG; Haemonetics), celite activated clotting time (ACT; Hemochron), and thrombin generation (TGA; Diapharma). Fresh blood was obtained from healthy volunteers under an IRB approved protocol (n=6 per group). Previous work from our lab (Jeske W, Thrombosis & Hemostasis Societies of North America, P57, 2018) identified a weaker potency of BH than PH when compared on an equigravimetric basis in pharmacopeial assays; however, equivalent potency could be obtained when BH was standardized against PH on a unitage basis. In this study, heparins were studied on both a gravimetric and a unitage basis for a comprehensive evaluation. The potency of the BH was determined by pharmacopeial compliant anti-Xa and anti-IIa chromogenic assays cross-referenced to the porcine USP Heparin Reference Standard.
Results: All results are depicted in Table 1. For the TEG, the R value, time of latency from start to initial fibrin formation, and the K value, time to achieve a defined clot strength due to thrombin generation and platelet activation, bovine heparin and porcine heparin did not demonstrate a significant difference in anticoagulant activity. The TEG angle, a measure of the speed of fibrin build-up and cross-linking (clot strengthening or rate of clot formation), and the maximum amplitude (MA) value, a function of the maximum of fibrin and platelet binding representing the strongest point of fibrin clot formation, also revealed no significant difference between bovine and porcine heparin anticoagulant activity. For the ACT, at concentrations of 10 µg/mL and 25 µg/mL, there were no statistically significant differences between BH and PH. For the TGA, measuring the time delay until the initiation of thrombin generation following tissue factor (TF) activation, the highest amount of thrombin generated after TF activation, and the total amount of thrombin generated after TF activation (AUC), showed a trend that PH was more potent than BH, but wide variation in the results did not allow for statistical differences to be identified.
Conclusion: The results of this investigation demonstrate that bovine heparin produces an equivalent anticoagulant activity as porcine heparin in the TEG, ACT, and TGA assay systems. The equivalent ACT results, in particular, were unexpected since gravimetric amounts of heparins were evaluated. As for all heparins, standardization of bovine heparin in accordance with the process used for porcine heparin will assure equivalent anticoagulant activity among bovine and porcine heparins in whole blood, plasma-based, and pharmacopeial assays. This study further demonstrates that the current assays used to monitor porcine heparin can be similarly used to monitor bovine heparin.
Disclosures
Walenga: Eurofarma: Research Funding.
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