Resourcing of Porcine Unfractionated and Low‐Molecular Weight Heparins from Ovine, and Bovine Sources

Abstract

IntroductionThe current shortage of porcine mucosal heparin supply warrants the need for exploring alternate sources to manufacture heparins. UFHs and LMWHs can also be derived from ovine and bovine sources, which could provide substitute for porcine derived anticoagulants. The purpose of this study is to compare the biologic properties of UFHs and LMWHs from porcine, ovine, and bovine sources at gravimetric concentrations.Materials and MethodsUSP standard (100 ug/ml), NIBSC (100 U/mL), APTT reagents (Triniclot), Spectrozymes Xa and IIa (Sekisui), Bovine Factor Xa (Enzyme Research, USA), Thrombin (Enzyme Research, USA), and blood bank plasma. The following agents were tested: porcine UFH (Medefil 13353), porcine LMWH (Enoxaparin 7L010), ovine UFH (lot# 1001), ovine LMWH (Enoxaparin lot# 404D), bovine UFH (GAG 25), and bovine LMWH (Enoxaparin lot# 003). All agents were diluted at a stock solution of 100 ug/mL in saline. Working dilutions were prepared at 100 ug/mL. Whole blood ACT was measured using a Hemochron instrument at a fixed concentration of 10 ug/mL and 25 ug/mL. Prothrombinase induced clotting time (PiCT), TT, aPTT, anti‐Xa, and anti‐IIa activities were measured in plasma supplemented with each of these agents at a concentration range of 0–10 ug/mL using the ACL Elite instrument. The USP potency assay was carried out using chromogenic antiprotease assays (Biophen Kits). The inhibitory concentrations at 50% (IC‐50) were measured using a purified antithrombin biochemical system employing a kinetic method. Thrombin generation inhibition assay was carried out using a CAT method (Diagnositca Stago, Paris, France).ResultsIn the ACT method, ovine UFH showed the highest value followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH. The PiCT results showed ovine UFH with the strongest anticoagulant activity followed by porcine UFH and bovine UFH. All three LMWH demonstrated comparable PiCT results and were significantly lower than their UFH counterparts. In the plasma supplementation studies, a similar trend was seen in the aPTT, TT, factor Xa, and factor IIa studies: ovine UFH demonstrated the strongest anticoagulant and antiprotease effects followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH. The IC‐50 values for the anti‐Xa ranged from 0.71–2.12 ug/mL for the UFH and from 2.23–3.92 for the LMWH, whereas the anti‐IIa values ranged from 3.91–8.37 ug/mL for the UFH and were greater than 10 ug/mL for the LMWH. Ovine UFH and LMWH had the lowest IC‐50 values for anti‐Xa in their respective categories while bovine UFH and LMWH had the highest. In the thrombin generation assays, the ovine UFH showed slightly stronger activities followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH in all three parameters including peak thrombin, lag time, and percent inhibition of area under the curve.