Resourcing of Heparin and Low Molecular Weight Heparins from Bovine, Ovine, and Porcine Origin. Studies to Demonstrate the Biosimilarities

Introduction:

The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. At the same time the demand for heparins has increased requiring alternate sources to obtain heparin and LMWH. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The pharmaceutical industry has already initiated programs to manufacture Bovine, Ovine, and Porcine heparins and depolymerized enoxaparin from these products some of which are currently in various phases of development. The purpose of this study is to compare 5 individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins.

Methods:

The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion method. A narrow range calibration method was used for comparing the molecular weight of heparin, whereas the EP method was used to cross-reference the molecular weight of enoxaparins. The anticoagulant potency was measured use clot based methods such as aPTT and Thrombin Time. Chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The interaction between AT and heparins and enoxaparin were investigated in a purified biochemical system, using AT supplemented buffered assay. Thrombin Generation inhibition studies were carried out using a flourometric method (Technoclone, Vienna, Austria). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry.

Results:

The molecular profile of the Bovine, ovine, and porcine heparins and enoxaparin were almost identical. In the clot based assays, such as PT and PTT, Porcine and Ovine heparin produce consistently comparable anticoagulant effects, which were stronger in comparison to the bovine derived heparins. In contrast, the enoxaparins derived from these three sources showed minimal differences. In the amidolytic anti Xa and IIa assays both ovine and porcine heparins produced similar inhibitory effects, whereas the bovine heparin exhibited lower activity. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of the Porcine and Ovine heparins ranged from 180 to 190u/mg, whereas the Bovine was found to be 130-140 u/mg. The anti-Xa - IIa ratio for the heparin were comparable. The ovine and porcine enoxaparin exhibited comparable potencies which ranged 94-110 u/mg whereas bovine enoxaparin was slightly lower 80-87 u/mg. However the antiXa and anti-IIa ratios were comparable. The AT mediated inhibition of factor Xa and anti-IIa was stronger with heparins in comparison to the enoxaparins. Similarly heparins produced stronger inhibition of thrombin generation in comparison to the enoxaparin. In the HIT screening there was no difference between the HIT responses in the heparins from different species. Similar results were obtained with enoxaparins.

Conclusions:

These studies show that while bovine, ovine and porcine heparins and enoxaparins exhibit comparable molecular profiles however in some of the functional assays bovine heparin and enoxaparin exhibited somewhat lesser potencies especially in the pharmacopeial assays. No differences were noted in the HIT antibody interactions among heparins and enoxaparins from different species. These studies demonstrate that ovine and porcine heparins are biosimilar and can be developed as such for clinical purposes. The bovine derived heparins exhibit slightly weaker potencies in functional assays despite comparable molecular profile. Potency adjustment for in vivo usage may be required to obtain comparable anticoagulant responses for the bovine heparin and enoxaparin.