PHYSICOCHEMICAL CHARACTERIZATION OF LOW-MOLECULAR-WEIGHT HEPARIN

Nuclear magnetic resonance spectroscopy (NMR), Raman spectroscopy, dyn amic light scattering (DLS), and high performance exclusion chromatogr aphy (HPEC) were used to characterize two different commercial prepara tions of low molecular weight (LMW) heparin, produced either by peroxi de cleavage or deaminative cleavage using nitrous acid. Proton NMR sho wed <2% contamination by dermatan sulfate in the material produced by deaminative cleavage using nitrous acid and <4% for the material produ ced by peroxide cleavage. The Raman spectra of the nitrous acid produc ed material showed an equivalent amount of O-sulfation to that in the material produced by peroxide, but about a 10% reduction in the conten t of N-sulfated glucosamine, as expected from the deamination reaction . DLS and HPEC indicated the presence of <0.2% of very high molecular weight/aggregate material for the peroxide preparation compared to 1% for the nitrous acid-prepared material. The weight average molecular w eight (M(w)) determined from HPEC was 5900 Da for the nitrous acid-pre pared material and 6850 Da for the peroxide-produced material. The num ber average molecular weight (M(n)) calculated from this data was 5200 Da for the nitrous acid preparation and 5300 Da for the peroxide-prod uced material. In addition, the nitrous acid-prepared material exhibit ed a much narrower size distribution of oligomeric species, as evidenc ed by the polydispersity (M(w)/M(n)) of 1.1 for the nitrous acid-prepa red material, as compared with a value of 1.3 for the peroxide-prepare d material. These studies demonstrate that significant differences bet ween preparations of LMW heparin can be resolved using these technique s. This is of critical importance in the design of quality assurance m ethods.