低分子肝素論文低分子肝素分子量測(cè)定用對(duì)照品的研制

1、低分子肝素論文：低分子肝素分子量測(cè)定用對(duì)照品的研制【中文摘要】低分子肝素(low molecular weight heparin, LMWH)是肝素經(jīng)化學(xué)或肝素酶降解或物理分級(jí)方法制備而成的肝素片段或組分,其平均分子量在8000以下,小于8000的組分不少于60%,抗FXa活性不低于70IU/mg,抗FXa/抗Flla不小于1.5。LMWH與肝素鈉主體結(jié)構(gòu)一致,是由艾杜糖醛酸或葡糖醛酸與氨基葡糖以及它們的硫酸化和乙?；苌锝M成的多糖鏈混合物為雙糖單位,以(14)糖苷鍵連接而成。近30年來(lái)臨床實(shí)踐證明,LMWH是優(yōu)良的抗凝抗血栓藥物,與肝素鈉相比抗血栓活性相當(dāng),但安全性更高。我國(guó)是世界肝素第

2、一生產(chǎn)大國(guó),但目前的狀況是80%以上的肝素產(chǎn)量以原料藥形式出口,出口的肝素原料藥經(jīng)加工后變成價(jià)格更高的LMWH制劑返銷回國(guó)。我國(guó)雖然已有十余個(gè)廠家生產(chǎn)LMWH制劑,但國(guó)內(nèi)LMWH質(zhì)量標(biāo)準(zhǔn)與國(guó)外相比明顯較低,主要滿足低分子肝素定義中分子量及活性的要求,未按工藝進(jìn)行分類,而不同工藝制備的LMWH有不同的末端結(jié)構(gòu),不同的工藝相關(guān)雜質(zhì),不同的平均分子量及分布,這些都影響低分子肝素的活性及體內(nèi)過(guò)程。因此,參照國(guó)外標(biāo)準(zhǔn),并結(jié)合國(guó)情,盡快提高國(guó)內(nèi)LMWH質(zhì)量標(biāo)準(zhǔn)十分必要。分子量及分布是LMWH質(zhì)量標(biāo)準(zhǔn)的的一個(gè)重要指標(biāo)。目前,還沒(méi)有國(guó)產(chǎn)LMWH分子量測(cè)定用對(duì)照品,只有歐洲藥典(European Pharmac

3、opoeia,EP)LMWH分子量測(cè)定用對(duì)照品,WHO LMWH分子量測(cè)定用對(duì)照品(International Reference Preparation LMWH for molecular weight calibration)和美國(guó)藥典(United States Pharmacopoeia,USP)依諾肝素鈉分子量測(cè)定用對(duì)照品。本課題致力于研制國(guó)產(chǎn)LMWH分子量對(duì)照品,研制了系列窄分布的LMWH分子量對(duì)照品和附帶一寬分布標(biāo)樣表的寬分布的LMWH分子量對(duì)照品,并將兩種對(duì)照品分別與WHO和USP對(duì)照品的測(cè)定結(jié)果進(jìn)行了對(duì)比。本研究取得的結(jié)果和結(jié)論有以下幾個(gè)方面：1本課題研制了系列窄分布的LM

4、WH分子量測(cè)定用對(duì)照品系列自制窄分布LMWH分子量對(duì)照品Mp分別為：1400,1850,2328,2863,5714,7768,10443。7個(gè)自制窄分布分子量對(duì)照品與USP依諾肝素鈉分子量對(duì)照品的校正曲線基本重合在一起,分別用自制窄分布對(duì)照品和USP依諾肝素鈉分子量對(duì)照品測(cè)定2個(gè)不同批次的依諾肝素鈉、USP依諾肝素鈉分子量系統(tǒng)適用性對(duì)照品以及EP LMWH分子量對(duì)照品的分子量,結(jié)果表明兩種對(duì)照品測(cè)定各樣品的Mw相差不大于50,證明制備的系列窄分布樣品可以作為L(zhǎng)MWH的分子量測(cè)定用對(duì)照品。分子量在6000以下的5個(gè)窄分布LMWH分子量測(cè)定用對(duì)照品(1400,1850,2328,2863,571

5、4)的制備方法是以依諾肝素鈉為原料,經(jīng)Biogel P10分離得到。Biogel p10分離依諾肝素鈉可見(jiàn)小分子量部分有鋸齒狀的峰,分別收集相應(yīng)的峰即可得到6000以下窄分布的系列對(duì)照品。6000以上的2個(gè)窄分布LMWH分子量測(cè)定用對(duì)照品(7768,10443)是以自制的(化學(xué)消除方法)MMWH (Mp8929)為原料,經(jīng)Biogel P10二次分離得到。本課題還嘗試了以SuperdexG75、SephadexG75、UltragelACA44為層析介質(zhì)分離肝素鈉,Sephadex G75為層析介質(zhì)分離55%分級(jí)醇沉的肝素鈉樣品和超濾分離在10kDa和5kDa間的組分,結(jié)果發(fā)現(xiàn)無(wú)論選擇何種原料

6、和層析介質(zhì)都無(wú)法得到類似分離依諾肝素鈉的鋸齒狀峰,且用以上方法得到的樣品產(chǎn)率和RI響應(yīng)值都較低。最后選擇產(chǎn)率及RI相應(yīng)值較高的MMWH為原料,經(jīng)Biogel P10二次分離后的組分作為700011100窄分布LMWH分子量對(duì)照品。該法制得的對(duì)照品與USP依諾肝素鈉窄分布分子量對(duì)照品比較峰仍較寬,采用SAX-HPLC和IPRP-HPLC進(jìn)一步分離自制700011100的窄分布LMWH分子量對(duì)照品,期望可以得到成分更單一的組分。結(jié)果,SAX-HPLC和IPRP-HPLC可以將依諾肝素鈉小分子量的組分進(jìn)一步分離成更單一的組分,但無(wú)法對(duì)分子量在700011100的組分進(jìn)一步分離。2研制了附帶一寬分布標(biāo)

7、樣表的寬分布LMWH分子量對(duì)照品。利用肝素鈉酶降解肝素鈉得到寬分布LMWH作為寬分布LMWH分子量對(duì)照品,附帶的寬分布標(biāo)樣表是由分子量從60018000的18個(gè)分子量與相應(yīng)的累積百分面積組成,根據(jù)寬分布標(biāo)樣表18個(gè)點(diǎn)的累積百分面積確定對(duì)應(yīng)寬分布對(duì)照品的保留時(shí)間,將保留時(shí)間與表中對(duì)應(yīng)分子量的對(duì)數(shù)值進(jìn)行3次曲線方程擬合,得到校正曲線并進(jìn)行分子量計(jì)算。自制寬分布分子量對(duì)照品與WHO分子量對(duì)照品UV和RI圖譜相似,兩種對(duì)照品得到的校正曲線幾乎重合在一起。用WHO LMWH分子量對(duì)照品和自制的寬分布分子量對(duì)照品分別測(cè)定7種LMWH肝素鈉分子量,兩者的測(cè)定結(jié)果不大50,說(shuō)明制備的樣品可以作為寬分布LMWH

8、分子量對(duì)照品測(cè)定各LMWH分子量。EP LMWH分子量對(duì)照品需要RI和UV聯(lián)用,專用GPC軟件,而本課題制備的兩種LMWH分子量對(duì)照品只需RI檢測(cè)器和普通GPC軟件,計(jì)算方法更簡(jiǎn)便。本課題成功研制了分子量從100011100的系列窄分布LMWH分子量對(duì)照品和附帶一寬分布標(biāo)樣表的寬分布分子量對(duì)照品,為L(zhǎng)MWH分子量測(cè)定用對(duì)照品的國(guó)產(chǎn)化奠定了基礎(chǔ)?！居⑽恼縇ow molecular weight heparin(LMWH) is produced by depolymeriation of heparin, via deaminative cleavage, or chemical or en

9、zymatic-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

10、 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-a

11、cetyl-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 dai

12、ly; 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 hepai

13、rn. 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.

14、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 ph

15、armacokinetics. 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 Europea

16、n 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 in

17、crease 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 11

18、100 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 140

19、0,1850,2328,2863,5714, 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

20、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

21、 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

22、 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 preparati

23、on 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

24、for molecular weight determination were established which lay a foundation for nationally made LMWH CRS.【關(guān)鍵詞】低分子肝素 分子量 分子量對(duì)照品 高效分子排阻法【英文關(guān)鍵詞】Low-molecular-weight hepairn molecular weight molecular weight calibrator high performance size exclusive chromatography【目錄】低分子肝素分子量測(cè)定用對(duì)照品的研制摘要10-13ABSTRACT13-1

25、5第一章 前言16-381 肝素鈉簡(jiǎn)介162 低分子肝素16-222.1 國(guó)外藥典對(duì)LMWH的收錄16-172.2 我國(guó)LMWH的概況172.3 生產(chǎn)LMWH常見(jiàn)的幾種化學(xué)降解方法17-212.4 LMWH分子量測(cè)定的重要性21-223 LMWH分子量的測(cè)定方法22-303.1 高效分子排阻色譜法22-243.2 聚丙烯酰胺凝膠電泳法24-263.3 多角度激光光散射儀法26-283.4 質(zhì)譜法28-293.5 核磁共振法29-304 LMWH幾種寬分布分子量對(duì)照品的概況30-334.1 EP LMWH分子量對(duì)照品30-314.2 WHO LMWH分子量對(duì)照品31-324.3 亞硝酸異戊酯降解

26、制備的LMWH分子量對(duì)照品32-334.4 化學(xué)消除法制備LMWH分子量對(duì)照品335 LMWH窄分布對(duì)照品的概況及肝素鈉分離純化的方法33-375.1 LMWH窄分布對(duì)照品概況33-345.2 肝素分離純化方法34-376 本課題擬解決的問(wèn)題37-38第二章 制備分子量在7000-11100范圍窄分布LMWH分子量對(duì)照品用原料38-481 材料38-392 方法39-412.1 分級(jí)醇沉法制備原料392.2 超濾法制備原料392.3 化學(xué)消除降解法制備原料39-413 結(jié)果41-453.1 分級(jí)醇沉制備原料的結(jié)果41-423.2 超濾法制備原料的結(jié)果423.3 化學(xué)消除降解制備原料的結(jié)果42-454 討論45-475