Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography.

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia.

The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium.

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0-102.8% and 101.6-102.8%, respectively), good intermediate precision (1.9-2.1% and 2.4-4.2%, respectively) and good reproducibility (4.0-4.8% and 3.6-6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.

Heparin identification test and purity test for OSCS in heparin sodium and heparin calcium by weak anion-exchange high-performance liquid chromatography.

Heparin sodium and heparin calcium, which are widely used as anti-coagulants, are known to potentially contain the natural impurity dermatan sulfate (DS). Recently serious adverse events occurred in patients receiving heparin sodium in the US, and a contaminant oversulfated chondroitin sulfate (OSCS) was found to be a cause of the events. To ensure the quality and safety of pharmaceutical heparins, there is need of a physicochemical identification test that can discriminate heparin from the heparin-related substances as well as a sensitive purity test for OSCS. Recently, HPLC with a strong-anion exchange column was proposed as the methods for identifying heparin and determination of OSCS in heparin sodium. Although this method is convenient and easy to perform, the only column suitable for this purpose is the Dionex IonPac AS11-HC column. In this study, we developed alternative identification test and test for OSCS in both heparin sodium and heparin calcium using a weak anion-exchange column. The identification test allowed for separation of heparin from the impurity DS and contaminant OSCS in a shorter time. The purity test provided enough sensitivity, specificity, linearity, recovery and repeatability for OSCS. We believe that our methods will be useful for quality control of pharmaceutical heparins.

Evaluation of Bacillus oleronius as a Biological Indicator for Terminal Sterilization of Large-Volume Parenterals.

In the production of large-volume parenterals in Japan, equipment and devices such as tanks, pipework, and filters used in production processes are exhaustively cleaned and sterilized, and the cleanliness of water for injection, drug materials, packaging materials, and manufacturing areas is well controlled. In this environment, the bioburden is relatively low, and less heat resistant compared with microorganisms frequently used as biological indicators such as Geobacillus stearothermophilus (ATCC 7953) and Bacillus subtilis 5230 (ATCC 35021). Consequently, the majority of large-volume parenteral solutions in Japan are manufactured under low-heat sterilization conditions of F0 <2 min, so that loss of clarity of solutions and formation of degradation products of constituents are minimized. Bacillus oleronius (ATCC 700005) is listed as a biological indicator in "Guidance on the Manufacture of Sterile Pharmaceutical Products Produced by Terminal Sterilization" (guidance in Japan, issued in 2012). In this study, we investigated whether B. oleronius is an appropriate biological indicator of the efficacy of low-heat, moist-heat sterilization of large-volume parenterals. Specifically, we investigated the spore-forming ability of this microorganism in various cultivation media and measured the D-values and z-values as parameters of heat resistance. The D-values and z-values changed depending on the constituents of large-volume parenteral products. Also, the spores from B. oleronius showed a moist-heat resistance that was similar to or greater than many of the spore-forming organisms isolated from Japanese parenteral manufacturing processes. Taken together, these results indicate that B. oleronius is suitable as a biological indicator for sterility assurance of large-volume parenteral solutions subjected to low-heat, moist-heat terminal sterilization.