低分子肝素论文低分子肝素分子量测定用对照品的研制.doc

低分子肝素论文：低分子肝素分子量测定用对照品的研制【中文摘要】低分子肝素(low molecular weight heparin, LMWH)是肝素经化学或肝素酶降解或物理分级方法制备而成的肝素片段或组分,其平均分子量在8000以下,小于8000的组分不少于60%,抗FXa活性不低于70IU/mg,抗FXa/抗Flla不小于1.5。LMWH与肝素钠主体结构一致,是由艾杜糖醛酸或葡糖醛酸与氨基葡糖以及它们的硫酸化和乙酰化衍生物组成的多糖链混合物为双糖单位,以(14)糖苷键连接而成。近30年来临床实践证明,LMWH是优良的抗凝抗血栓药物,与肝素钠相比抗血栓活性相当,但安全性更高。我国是世界肝素第一生产大国,但目前的状况是80%以上的肝素产量以原料药形式出口,出口的肝素原料药经加工后变成价格更高的LMWH制剂返销回国。我国虽然已有十余个厂家生产LMWH制剂,但国内LMWH质量标准与国外相比明显较低,主要满足低分子肝素定义中分子量及活性的要求,未按工艺进行分类,而不同工艺制备的LMWH有不同的末端结构,不同的工艺相关杂质,不同的平均分子量及分布,这些都影响低分子肝素的活性及体内过程。因此,参照国外标准,并结合国情,尽快提高国内LMWH质量标准十分必要。分子量及分布是LMWH质量标准的的一个重要指标。目前,还没有国产LMWH分子量测定用对照品,只有欧洲药典(European Pharmacopoeia,EP)LMWH分子量测定用对照品,WHO LMWH分子量测定用对照品(International Reference Preparation LMWH for molecular weight calibration)和美国药典(United States Pharmacopoeia,USP)依诺肝素钠分子量测定用对照品。本课题致力于研制国产LMWH分子量对照品,研制了系列窄分布的LMWH分子量对照品和附带一宽分布标样表的宽分布的LMWH分子量对照品,并将两种对照品分别与WHO和USP对照品的测定结果进行了对比。本研究取得的结果和结论有以下几个方面：1本课题研制了系列窄分布的LMWH分子量测定用对照品系列自制窄分布LMWH分子量对照品Mp分别为：1400,1850,2328,2863,5714,7768,10443。7个自制窄分布分子量对照品与USP依诺肝素钠分子量对照品的校正曲线基本重合在一起,分别用自制窄分布对照品和USP依诺肝素钠分子量对照品测定2个不同批次的依诺肝素钠、USP依诺肝素钠分子量系统适用性对照品以及EP LMWH分子量对照品的分子量,结果表明两种对照品测定各样品的Mw相差不大于50,证明制备的系列窄分布样品可以作为LMWH的分子量测定用对照品。分子量在6000以下的5个窄分布LMWH分子量测定用对照品(1400,1850,2328,2863,5714)的制备方法是以依诺肝素钠为原料,经Biogel P10分离得到。Biogel p10分离依诺肝素钠可见小分子量部分有锯齿状的峰,分别收集相应的峰即可得到6000以下窄分布的系列对照品。6000以上的2个窄分布LMWH分子量测定用对照品(7768,10443)是以自制的(化学消除方法)MMWH (Mp8929)为原料,经Biogel P10二次分离得到。本课题还尝试了以SuperdexG75、SephadexG75、UltragelACA44为层析介质分离肝素钠,Sephadex G75为层析介质分离55%分级醇沉的肝素钠样品和超滤分离在10kDa和5kDa间的组分,结果发现无论选择何种原料和层析介质都无法得到类似分离依诺肝素钠的锯齿状峰,且用以上方法得到的样品产率和RI响应值都较低。最后选择产率及RI相应值较高的MMWH为原料,经Biogel P10二次分离后的组分作为700011100窄分布LMWH分子量对照品。该法制得的对照品与USP依诺肝素钠窄分布分子量对照品比较峰仍较宽,采用SAX-HPLC和IPRP-HPLC进一步分离自制700011100的窄分布LMWH分子量对照品,期望可以得到成分更单一的组分。结果,SAX-HPLC和IPRP-HPLC可以将依诺肝素钠小分子量的组分进一步分离成更单一的组分,但无法对分子量在700011100的组分进一步分离。2研制了附带一宽分布标样表的宽分布LMWH分子量对照品。利用肝素钠酶降解肝素钠得到宽分布LMWH作为宽分布LMWH分子量对照品,附带的宽分布标样表是由分子量从60018000的18个分子量与相应的累积百分面积组成,根据宽分布标样表18个点的累积百分面积确定对应宽分布对照品的保留时间,将保留时间与表中对应分子量的对数值进行3次曲线方程拟合,得到校正曲线并进行分子量计算。自制宽分布分子量对照品与WHO分子量对照品UV和RI图谱相似,两种对照品得到的校正曲线几乎重合在一起。用WHO LMWH分子量对照品和自制的宽分布分子量对照品分别测定7种LMWH肝素钠分子量,两者的测定结果不大50,说明制备的样品可以作为宽分布LMWH分子量对照品测定各LMWH分子量。EP LMWH分子量对照品需要RI和UV联用,专用GPC软件,而本课题制备的两种LMWH分子量对照品只需RI检测器和普通GPC软件,计算方法更简便。本课题成功研制了分子量从100011100的系列窄分布LMWH分子量对照品和附带一宽分布标样表的宽分布分子量对照品,为LMWH分子量测定用对照品的国产化奠定了基础。【英文摘要】Low molecular weight heparin(LMWH) is produced by depolymeriation of heparin, via deaminative cleavage, or chemical or enzymatic-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-factora 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 (14)bonds1.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 factora 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.【关键词】低分子肝素 分子量 分子量对照品 高效分子排阻法【英文关键词】Low-molecular-weight hepairn molecular weight molecular weight calibrator high performance size exclusive chromatography【目录】低分子肝素分子量测定用对照品的研制摘要10-13ABSTRACT13-15第一章 前言16-381 肝素钠简介162 低分子肝素16-222.1 国外药典对LMWH的收录16-172.2 我国LMWH的概况172.3 生产LMWH常见的几种化学降解方法17-212.4 LMWH分子量测定的重要性21-223 LMWH分子量的测定方法22-303.1 高效分子排阻色谱法22-243.2 聚丙烯酰胺凝胶电泳法24-263.3 多角度激光光散射仪法26-283.4 质谱法28-293.5 核磁共振法29-304 LMWH几种宽分布分子量对照品的概况30-334.1 EP LMWH分子量对照品30-314.2 WHO LMWH分子量对照品31-324.3 亚硝酸异戊酯降解制备的LMWH分子量对照品32-334.4 化学消除法制备LMWH分子量对照品335 LMWH窄分布对照品的概况及肝素钠分离纯化的方法33-375.1 LMWH窄分布对照品概况33-345.2 肝素分离纯化方法34-376 本课题拟解决的问题37-38第二章 制备分子量在7000-11100范围窄分布LMWH分子量对照品用原料38-481 材料38-392 方法39-412.1 分级醇沉法制备原料392.2 超滤法制备原料392.3 化学消除降解法制备原料39-413 结果41-453.1 分级醇沉制备原料的结果41-423.2 超滤法制备原料的结果423.3 化学消除降解制备原料的结果42