USP referenced potency evaluation of bovine heparins utilizing currently available assays

Abstract

IntroductionHeparin is the most widely used anticoagulant drug for medical, surgical and interventional usage. The currently used heparin is primarily obtained from porcine intestinal mucosa. More recently, the reintroduction of bovine heparin is being considered for similar indications. The bovine heparins are mostly obtained from mucosal tissues.AimThe purpose of this study is to cross‐reference the anticoagulant and USP potency of the currently developed bovine mucosal heparins.Materials & MethodsSeveral batches (10–20) of bovine heparins were obtained from two separate manufacturers (A & B) in powdered form. Porcine mucosal heparin was obtained from Medefil Inc. Glendale Heights, IL. USP heparin reference standard (LOT# FOI187) was obtained from US Pharmacopeia Bethesda, MD. Pooled blood bank plasma was purchased from the blood bank at Loyola University Medical Center. Biophen Heparin anti‐Xa and anti‐IIa assays (Aniara, Mason, OH) were used to measure the potency of various heparins against the USP standard. Purified antithrombin supplemented system was used to study the antiprotease activities of these heparins in terms of IC50. For anticoagulant assays, the Triniclot aPTT reagent (Tcoag, Wicklow, Ireland) was used.ResultsIn comparison to the porcine mucosal heparin, the bovine heparins from both sources exhibited considerably lower USP referenced anti‐Xa and anti‐IIa activites. Heparins from manufacturer A exhibited potencies of 145 ± 3 anti‐Xa units/mg and 139 ± 7 anti‐IIa units/mg with an anti‐Xa/anti‐IIa ratio of 1.05. In the case of manufacturer B, heparins exhibited potencies of 141 ± 11 anti‐Xa units/mg and 142 ± 12 anti‐IIa units/mg. Potencies determined by aPTT clotting assay were found to be higher for heparins from both manufacturers (A: 149 ± 4 U/mg; B:(154 ± 13 U/mg) compared to those determined by amidolytic assay. The products from manufacturer B showed wider lot‐to‐lot variation in potency. In the purified antithrombin systems the IC50 of bovine heparins (3.7 ± 0.2 ug/ml for Xa and 0.17 ± 0.1 ug/ml for IIa) were much higher in comparison to the IC50 values of porcine heparin (1.23 ug/ml for Xa and 0.16 ug/ml for IIa).ConclusionsThese studies clearly demonstrate that the currently available USP standard can be used to cross reference the potency of bovine mucosal heparins from various vendors. Furthermore, the bovine heparins exhibit lower potency in comparison to porcine mucosal heparin in both the amidolytic and clot‐based assays. The potency calculated using the plasma‐based clotting assays, such as aPTT, provides higher potency for the bovine heparins in comparison to the amidolytic methods. The marked differences in the IC50 of the Xa inhibition clearly indicates that the bovine heparins differ considerably in the lower molecular weight range in terms of antithrombin binding components, whereas the higher molecular weight components with antithrombin affinity may be similar.