Development of an Industrial Processing Method for Removing Contaminating Mucopolysaccharides from Low Molecular Weight Heparin Preparations

We have developed a simple method for removing mucopolysaccharide (MS) impurities from low molecular weight heparin (LMWH) preparations. Crude dalteparin sodium (DN-C) containing high molecular weight impurities such as sodium salts of over-sulfated chondroitin sulfate (OSH) and dermatan sulfate (DSH) was used as a model of LMWH preparations. In preliminary experiments, the sensitivities of OSH and DSH to ethanol were quite different, even though their molecular weights are similar. A two-step ethanol precipitation was used to remove these contaminants. Upon the first treatment, OSH was removed as a precipitate, and upon the second treatment, DSH was recovered, in the supernatant. LMWH was concentrated in the precipitate following the second treatment. The contaminants were detected easily by nitrous acid de-polymerization followed by gel permeation HPLC. In contrast, it was difficult to detect small amounts of impurities by H-1-NMR. We also tried purifying commercial DN products (DNG 1-3) to confirm the efficacy of this methodology and concluded that the two-step ethanol precipitation of LMWH, followed by purity analysis by gel permeation HPLC after nitrous acid de-polymerization of MSs, holds promise for the manufacture of high-quality LMWH preparations.