Physiochemical characterization of low molecular weight heparin

Nuclear magnetic resonance spectroscopy (NMR), Raman spectroscopy, dynamic light scattering (DLS), and high performance exclusion chromatography (HPEC) were used to characterize two different commercial preparations of low molecular weight (LMW) heparin, produced either by peroxide cleavage or deaminative cleavage using nitrous acid. Proton NMR showed < 2% contamination by dermatan sulfate in the material produced by deaminative cleavage using nitrous acid and < 4% for the material produced by peroxide cleavage. The Raman spectra of the nitrous acid produced material showed an equivalent amount of O-sulfation to that in the material produced by peroxide, but about a 10% reduction in the content 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 weight (Mw) determined from HPEC was 5900 Da for the nitrous acid-prepared material and 6850 Da for the peroxide-produced material. The number average molecular weight (Mn) calculated from this data was 5200 Da for the nitrous acid preparation and 5300 Da for the peroxide-produced material. In addition, the nitrous acid-prepared material exhibited a much narrower size distribution of oligomeric species, as evidenced by the polydispersity (Mw/Mn) of 1.1 for the nitrous acid-prepared material, as compared with a value of 1.3 for the peroxide-prepared material. These studies demonstrate that significant differences between preparations of LMW heparin can be resolved using these techniques. This is of critical importance in the design of quality assurance methods.