Preparation Of The Molecular Mass Calibration Reference Standard For Molecular Weight Hepairn

Low molecular weight heparin(LMWH) is produced by depolymeriation of heparin, via deaminative cleavage, or chemical or enzymaticβ-elimation or by oxidative depolymeriaztion. LMWHs are defined as heparin salts having an average molecular weight of less than 8000 Da and for which at least 60% of all chains have a molecular weight less than 8000 Da. Its potency is not less than 70IU of anti-factor Xa activity per miligram and the ratio of anti-factor Xa activity to anti-factorⅡa activity is not less than 1.5. The backbone structure of LMWH is the same as hepairn composed of alternate sequences of differently sulfated residues of uronic acid (a-L-iduronic acid or (3-D-glucuronic) and gulcosamine(a-D-glucosamine or N-acetyl-a-D-gucosamine) linked by (1→4)bonds[1].LMWH had been introduced into clinical practice some 30 years ago. A number of distinct advantages that LMWH offer over hepairn, such as high anti-factor Xa than factorⅡa activity; bioavailability approaching 100%; leading to administration once or twice daily; lesser interaction with heparin-binding proteins. Therefore, due to its greater efficiency, less hemorrhagic effects, and improved convenience, LMWHs are now the preferred treatment in the field of thrombosis and hemostasis as compared with hepairn.China is the main contry of that produces hepairn. The current situation is that more than 80% yield of heparin was exported as API, then LMWH preparations at much higher price reselled to China. There are already more than 10 companies that product LMWH preparations in our contry, but the quality standard is inferior to international standard. The basic requirments of LMWH limit to molecular weight and potency, not classified according to their process. It has demonstrated that LMWHS produced by different methods differ in their molecular weight, end structures and process-related impurites which play roles in their potencies and their pharmacokinetics. Therefore, the domestic quality standard of LMWH should be impoved urgently. One of the most important aspect in LMWH quality standard is the molecular weight. There is no domestic LWMH calibration reference standard(CRS) for its molecular weight calibration. Untill now, only European Pharmacopoeia(EP) LMWH CRS, WHO LMWH CRS and United States Pharmacopoeia(USP) CRS for enoxaparin sodium molecular weight calibrations. Development standard for quality control of LMWH molecular weight will no doubt boost the domestic pharmaceutical companies to improve their product quality and increase their market competiveness. In the present study, both narrow and broad distribution molecular weight CRS of LMWH have been successfully prepared. The results and conclusions of the studies are the following:1 Seven narrow references for LMWH molecular weight calibration range from 1400 to 11100 in accordance with USP enoxaprin sodium molecular weight calibrations were prepared. Skewed peaks 1-7 appeared when Enoxaparin sodium was separated by Biogel P10. Peaks were collected respectly, peakl-4 and peak 7 were chosen as narrow molecular weight calibrators whose moleuclar weights are 1400,1850,2328,2863,5714, calibrated by both USP-HPSEC. MMWH was prepared by mild chemical P-elimination (Mp8929). MMWH was secondly separated on Biogel P10, the corresponding fraction as the narrow molecular weight calibrators was collected. The molecuar weight are 7768,10443 which were determined by USP-HPLC. The 7 narrow calibrators moleuclar weight also determined by HPSEC-MALLS compared with USP-HPSEC,with relative deviation range from 2% to 10%. The molecular weight of two batches of Enoxparin sodium, USP enoxaparin sodium CRS for system suitabilty and EP LMWH CRS were tested by USP CRS and 7self-made narrow standards. The differece of molecular weight result is less than 50 which demonstrated 7 self-made narrow standards for LMWH molecular weight calibration are feasible.2 Broad distribution molecular weight calibrator was prepared by partial degraded of hepairn with hepairnase I and a calibration table was provided by the WHO-HPSEC. The UV, RI detector and its calibration curve are similar to WHO LMWH calibrator. Using this calibrator and WHO calibrator we have measured the molecular weight of 7 LMWHs, the results deviation was within 50Da. The date demonstrated this preparation could be used as broad molecular weight calibrator for LMWH.EP CRS for LMWH calibration requires both RI and UV detectors, and special GPC software whereas the narrow and broad calibrators prepared in this project need only RI detector and the ordinary GPC software. The narrow and broad LMWH CRS for molecular weight determination were established which lay a foundation for nationally made LMWH CRS.