Structural analysis of bovine derived heparins

Abstract

The differences in heparins from different sources become much more subtle as they are depolymerized into low molecular weight heparin (LMWH). These differences are often sufficiently small so that they require principle component analysis (PCA) to determine. Differences in the reducing end and the non-reducing end structures in heparin determined by mass spectrometry (MS) as well as differences in the glucosamine and uronic acid residues determined by nuclear magnetic resonance spectroscopy (NMR) were selected for PCA. Using PCA it was possible to link parent heparin starting material to its daughter LMWH. This analysis demonstrated the lower variation between LMW bovine and LMW porcine heparins than between bovine and porcine heparins. This lower variation afforded the LMW bovine heparin similar anti-Xa and anti-IIa activity comparable to commercial heparin.

Bovine heparin is characteristically different than porcine intestinal heparin. These differences include sulfation, molecular weight properties, activity, structure, and shape. Bovine lung heparin has a higher amount of GlcNY6S (where Y can represent Ac or S) while the amount of GlcNY6S is much lower in bovine intestinal heparin. All heparins have high amounts of trisulfated TriS disaccharide but the level of TriS is in lower in bovine intestinal heparin. The amount of NS2S is much higher in bovine intestinal heparin than in bovine lung and porcine intestinal heparins. The average molecular weight of bovine intestinal heparin is similar to porcine intestinal heparin but the molecular weight of bovine lung heparin is much lower. The activities of bovine tissue heparins were comparable to, but lower than, the activity of porcine intestinal heparin.

Small differences in counterfeit heparin, i.e. blended porcine and bovine heparins were next examined. Porcine heparin and bovine heparin of similar molecular weights to obtain a bovine heparin counterfeit drug of enhanced activity. Such counterfeit blends are undetectable by current methods of analysis. Diffusion ordered spectroscopy (DOSY) method for analysis was developed. DOSY exploits differences in the molecular shape of bovine heparin and porcine heparin and achieved partial separation in the diffusion dimension. Additional spectra for component resolution (SCORE) analysis were used to demonstrate detection and identification of blend mixture.

The harsh purification process used to prepare heparin leaves the heparin product largely unaffected except for its reducing end tetrasaccharide linkage region. GlyserineAc is present in porcine and bovine heparin but is absent in LMW heparin. We hypothesized that the peracetic acid bleaching adds an O-acetyl group that is selectively lost during β-elimination in the LMWH production process. The tetrasaccharide composition of bovine and porcine heparin is similar as are different batches from the same supplier. This emphasizes how similar processing results in similar heparin regardless of whether bovine or porcine is used.