PB0577 - Heparin Potency Assays – reagents can cause differences in potency assignment

Background: Potency determination of heparin is well defined in both the respective United States and European Pharmacopeia monographs. The precise quantities of reagents required for the antithrombin dependent anti-IIa assay (aIIa) and the antithrombin dependent anti-Xa assay (aXa) are clearly stated. The estimates by aIIa are used for labelled potencies and the ratios of aIIa/aXa which must be within the value of 0.9 and 1.1 for heparin are used as a bio-identity check. Testing carried out in our laboratory following a change in reagents generated ratios outside this range for some heparin samples, which indicate that reagent source may influence activity determinations.

Aims: To investigate the influence of different reagent combinations on activity determination of heparin samples.

Methods: The pharmacopoeial monograph methods for aIIa and aXa were employed with different reagents to determine activity of porcine and bovine heparin samples relative to the current International Standard for Unfractionated Heparin (IS).

Results: When assayed against the IS, three different antithrombin reagents gave different activities for bovine heparin (see figure 1a) – for aIIa p = 0.033, 0.953, 0.206, for aXa p = 0.065, < 0.001, < 0.001. There were no differences due to antithrombin on activity determinations of porcine heparin samples (see figure 1b).

Conclusion(s): The source of antithrombin used can influence the activity determination of bovine heparin by both aIIa and aXa assays, whilst there was no observable impact on potencies of porcine heparins. This observation may have some implications for the potency assignment of bovine unfractionated heparin.