WO2019000335A1 - Standard library of low-molecular-weight heparin, dalteparin sodium, and preparation method thereof - Google Patents

Standard library of low-molecular-weight heparin, dalteparin sodium, and preparation method thereof Download PDF

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Publication number
WO2019000335A1
WO2019000335A1 PCT/CN2017/090902 CN2017090902W WO2019000335A1 WO 2019000335 A1 WO2019000335 A1 WO 2019000335A1 CN 2017090902 W CN2017090902 W CN 2017090902W WO 2019000335 A1 WO2019000335 A1 WO 2019000335A1
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precipitate
liquid
heparin
molecular weight
sodium
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PCT/CN2017/090902
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French (fr)
Chinese (zh)
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周霞
雷晓刚
郭维
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辰欣药业股份有限公司
山东辰中生物制药有限公司
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Priority to PCT/CN2017/090902 priority Critical patent/WO2019000335A1/en
Publication of WO2019000335A1 publication Critical patent/WO2019000335A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions

Definitions

  • the invention relates to the field of biomedicine, in particular to a low molecular weight heparin sodium heparin standard library and a preparation method thereof.
  • Heparin (unfractionated heparin, unfractionated heparin) is a highly sulfated glycosaminoglycan that has anticoagulant effects both in vivo and in vitro.
  • unfractionated heparin has defects such as low bioavailability, large side effects, and excessive frequency of administration, and its replacement product
  • low molecular weight heparin such as Dartare is not only superior to unfractionated heparin, but also has high bioavailability and long half-life in vivo. , bleeding tendency is small and so on.
  • the anticoagulant activity of heparin is divided into two major categories: antithrombotic activity (FXa) and anticoagulant (FIIa) activity.
  • anti-FXa activity and anti-FIIa activity are essential, antithrombotic effect.
  • the measure is expressed by the anti-FXa/FIIa value, and the larger the value, the stronger the antithrombotic effect and the smaller the bleeding tendency.
  • Low molecular Dartare is a kind of low molecular weight heparin obtained by nitrite degradation. It is an upgraded product of unfractionated heparin. The relative average molecular weight is between 5600 and 6400 Da, and the average value is 6000 Da.
  • Low-molecular Dalteparin Sodium is an unfractionated heparin that reacts with nitrite at specific temperature and pH conditions to react N-glucosamine sulfate in unfractionated heparin to remove HSO 4 - form -NH 2 , -NH 2 undergoes diazotization reaction with nitrite, cleavage of glycosidic bond while releasing N 2 , electron transfer, condensed ring to form 2,5-dehydromannuraldehyde, and the latter undergoes reducing sugar or dehydrogenation to form mannose alcohol. It has better antithrombotic activity, less bleeding risk, and lower platelet impact than heparin products.
  • the prepared low molecular weight heparin sample has disadvantages such as insufficient purity, inconsistent molecular weight distribution, and excessive residual impurities.
  • the use of low molecular weight heparin sodium heparin to form a standard library that can be used for research requires high quality, high quality low molecular weight heparin sodium, which is a problem that cannot be solved by current processes.
  • a first object of the present invention is to provide a low molecular weight heparin sodium heparin standard for the above-mentioned deficiencies of the prior art.
  • the preparation method of the library Compared with other low molecular weight heparin sodium, the high quality low molecular weight heparin sodium heparin standard library prepared by this method has very good stability and antithrombotic activity.
  • a second object of the present invention is to provide a low molecular weight heparin sodium heparin standard library prepared by the preparation method of the above low molecular weight heparin sodium heparin standard library, which has high purity, concentrated molecular weight distribution and good antithrombotic activity.
  • a method for preparing a low molecular weight heparin sodium heparin standard library comprises the following steps:
  • Enzymatic hydrolysis Add 10 to 15 times the amount of water (mass ratio) to the crude heparin sodium, dissolve the raw material solution, adjust the temperature of the raw material liquid to 35 ° C to 39 ° C, add the complex enzyme, and stir the reaction for 10 to 15 hours. Precipitate, filter the supernatant, and collect the filtrate;
  • step 3 ultrafiltration and impurity removal: using the ultrafiltration membrane with molecular weight cut off of 3,000 Da to 5,000 Da, the purified liquid obtained in step 2) is subjected to tangential flow circulation ultrafiltration, and circulating ultrafiltration for 5 to 10 hours to collect the retentate;
  • Alcohol precipitation and impurity removal the retentate alcohol is removed and the precipitate is dried and weighed;
  • step 4 Degradation: 7-8 times of water is added to the precipitate obtained in step 4), the pH of the solution is adjusted to 2.0-4.0 with hydrochloric acid solution, the temperature of the solution is adjusted to 15-30 ° C, and an appropriate amount of sodium nitrite is added. Agitating the reaction for 90 to 120 minutes; the sodium nitrite is added in an amount of from 2% to 3% by mass of the precipitate obtained in the step 4);
  • step (1) is 0.9% to 1.1% of the mass of the precipitate; the mass to volume ratio of sodium chloride to the feed liquid is 0.9% to 1.1%;
  • step 8) ultrafiltration purification: the solution obtained in step 7) is ultrafiltered with a membrane of 5000 Da to 6000 Da, and the retentate is collected; the retentate is ultrafiltered through a membrane of 0.22 ⁇ m to obtain a filtrate, and the pH of the filtrate is adjusted to 6.4 to 7.4;
  • the complex enzyme comprises papain, ribonuclease II and deoxyribonuclease I, the amount of papain is 1% to 2% of the weight of the crude heparin sodium, and the amount of ribonuclease II is the weight of the crude heparin sodium. 0.5% to 1%, The amount of DNase I is from 0.5% to 1% by weight of the crude heparin sodium.
  • step 4) and step 7 the alcohol precipitation operation can be carried out by the alcohol precipitation method well known in the prior art, but in order to obtain a better purification effect, the following specific methods are preferably implemented:
  • Step 4 When the alcohol is removed, the 0.6 to 0.8 volume of methanol is added to the retentate obtained in the step 3), and the precipitate is precipitated for 14 to 18 hours to separate the precipitate; the precipitate is dissolved in water, and 1 is added to the solution. ⁇ 1.2 times the volume of methanol, precipitated for 14 to 18 hours, the precipitate was separated, and the precipitate was vacuum dried and weighed;
  • Step 7) The specific process of alcohol precipitation: adjust the pH of the feed solution to 6-8 with hydrochloric acid solution, add appropriate amount of sodium chloride and 2 to 3 volumes of ethanol, stir for 20 to 30 minutes, and then settle the precipitate 8 to 10 In the hour, the supernatant is removed; the mass to volume ratio of sodium chloride to the feed liquid is 1% to 3%.
  • the alcohol liquid is preferably a methanol solution or an ethanol solution.
  • a low molecular weight heparin sodium heparin standard library is prepared by the preparation method of the low molecular weight heparin heparin sodium standard library.
  • the molecular weight of the low molecular weight heparin sodium heparin standard library is 5800Da to 6200Da, and the molecular weight distribution is ⁇ 1600.
  • the ratio is ⁇ 40.0%; the ratio of > 4500 is ⁇ 11.0%.
  • the preparation method of the low molecular weight heparin sodium heparin standard library of the invention has the following outstanding
  • the average molecular weight of the low molecular weight heparin sodium heparin standard library prepared by the method of the invention is 5800-6200, the molecular weight distribution: ⁇ 1600 ratio ⁇ 40.0%; > 4500 ratio ⁇ 11.0%; anti-FXa/FIIa> 30, anti-FXa titer: 195 ⁇ 210IU / mg, anti-FIIa titer: ⁇ 5.0IU / mg, compared with other low molecular weight heparin sodium, has very good stability and antithrombotic activity, product quality beyond the European Pharmacopoeia standards It is expected to become a new generation of heparin antithrombotic drugs.
  • the low molecular weight heparin sodium heparin standard library provided by the invention has a molecular weight of 5800 Da to 6200 Da, a molecular weight distribution: ⁇ 1600 ratio ⁇ 40.0%; > 4500 ratio ⁇ 11.0%. It has the advantages of high purity, concentrated molecular weight distribution, and meets the stringent requirements for standard products in scientific research activities. It is very suitable as a standard in scientific research activities. use.
  • Enzymatic hydrolysis Weigh 20 mg of crude heparin sodium, add 240 mg of water to it, dissolve the raw material solution; adjust the temperature of the raw material solution to 37 ° C, add 0.2 mg of papain, 0.1 mg of ribonuclease II and deoxyribonuclease I, respectively. 0.1 mg, the reaction was stirred for 12 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
  • control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 9.5-10 with 20% (w/v) sodium hydroxide solution, add 2.4 ⁇ l of hydrogen peroxide, and stir the reaction. After 7 hours, the first purified liquid was obtained.
  • the first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 4,000 Da, and subjected to ultrafiltration for 7 hours to collect 63.2 ⁇ l of the first retentate.
  • step 5 Degradation: 120 mg of water was added to the first precipitate obtained in step 4), the pH was adjusted to 2.0 to 4.0 with 2 mol/L hydrochloric acid solution, the temperature was adjusted to 15 ° C, 0.4 mg of sodium nitrite was added, and the reaction was stirred for 90 minutes. , the first liquid is obtained.
  • Alkaline oxygen purification adjust the pH of the fourth liquid to 9.5-10 with 20% (w/v) sodium hydroxide solution, add 1.5% of the fourth liquid volume of hydrogen peroxide, and stir at 25 °C. The reaction was carried out for 7 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth liquid solution is adjusted to 6.5-7 with a 1 mol/L hydrochloric acid solution, and 1.5% (w/v) of the fifth liquid volume of NaCl and 2.5 times of the fifth liquid volume of ethanol are added thereto. After stirring for 25 minutes, the precipitate was allowed to stand for 9 hours, and the supernatant was removed to obtain a third precipitate. The third precipitate was dissolved in 60 mg of water to obtain a second purified solution.
  • Enzymatic hydrolysis Weigh 40 mg of crude heparin sodium, add 400 mg of water to it, dissolve the raw material solution; adjust the temperature of the raw material solution to 37 ° C, and add papain 0.8 mg, ribonuclease II 0.2 mg and deoxyribonuclease I0, respectively. .2 mg, the reaction was stirred for 12 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
  • control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 9.5-10 with 20% (w/v) sodium hydroxide solution, add 5 ⁇ l of hydrogen peroxide, and stir the reaction 7 In hours, the first purified liquid was obtained.
  • the first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 4000 Da, and subjected to ultrafiltration for 7 hours, and 140 ⁇ l of the first retentate was collected.
  • step 5 Degradation: add 240 mg of water to the first precipitate obtained in step 4), adjust the pH to 2.0 to 4.0 with 2 mol/L hydrochloric acid solution, adjust the temperature to 30 ° C, add 0.8 mg of sodium nitrite, and stir the reaction for 120 minutes. , the first liquid is obtained.
  • Enzymatic hydrolysis 40 mg of crude heparin sodium was weighed, 400 mg of water was added thereto, and the raw material liquid was dissolved; the temperature of the raw material liquid was adjusted to 35 ° C, and papain 0.4 mg, ribonuclease II 0.2 mg, and deoxyribonuclease I0 were respectively added. .2 mg, the reaction was stirred for 10 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
  • control step 1) to obtain a temperature of the first filtrate to 40 ° C, adjust the pH of the first filtrate to 9.0 to 10.0 with 20% (w/v) sodium hydroxide solution, add 4 ⁇ l of hydrogen peroxide, and stir the reaction for 6 hours.
  • the first purified liquid was obtained.
  • the first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 3000 Da, and subjected to ultrafiltration for 5 hours, and 140 ⁇ l of the first retentate was collected.
  • Alcohol precipitation 84 ⁇ l of methanol was added to the first retentate obtained in step 3), and the precipitate was precipitated for 14 hours to separate a primary precipitate; the primary precipitate was dissolved in water to 140 ⁇ l, and 140 ⁇ l of methanol was added to precipitate for 14 hours. The first precipitate was isolated and the first precipitate was dried in vacuo and weighed (32 mg).
  • step 5 Degradation: Add 224 mg of water to the first precipitate obtained in step 4), adjust the pH to 2.0-3.0 with 2 mol/L hydrochloric acid solution, adjust the temperature to 15 ° C, add 0.64 mg of sodium nitrite, and stir the reaction for 90 minutes. , the first liquid is obtained.
  • the second filtrate was filtered, and 3 volumes of medicinal ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 12 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 240 ⁇ l, stirred to dissolve, and ultraviolet irradiation was carried out for 20 minutes to obtain a fourth liquid.
  • Alkaline oxygen purification adjust the pH of the fourth liquid to 9.0-9.5 with 20% (w/v) sodium hydroxide solution, and add 1% of the fourth liquid volume of hydrogen peroxide at a temperature of 20 °C. The reaction was stirred for 6 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth solution is adjusted to 6.0 to 6.5 with a 1 mol/L hydrochloric acid solution, and 1% (w/v) of the fifth solution volume of NaCl and 2 times of the fifth solution volume of ethanol are added thereto. After stirring for 20 minutes, the precipitate was allowed to stand for 8 hours, and the supernatant was removed to obtain a third precipitate, and the third precipitate was dissolved in 120 mg of water to obtain a second purified solution.
  • the second purification liquid obtained in the step 7) is subjected to ultrafiltration through a membrane of 5000 Da, and the second retentate is collected: the second retentate is ultrafiltered through a membrane of 0.22 ⁇ m to obtain a third filtrate, and the third adjustment is performed.
  • the pH of the filtrate was adjusted to 6.4-7.
  • Lyophilization The third filtrate will be lyophilized after adjusting the pH to obtain a low molecular weight heparin to a heparin sodium standard library of 17 mg.
  • Enzymatic hydrolysis Weigh 40 mg of crude heparin sodium, add 600 mg of water to it, dissolve the raw material solution; adjust the temperature of the raw material solution to 39 ° C, and add papain 0.8 mg, ribonuclease II 0.4 mg and deoxyribonuclease I0, respectively. .4 mg, the reaction was stirred for 15 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
  • control step 1) to obtain a temperature of the first filtrate to 50 ° C, adjust the pH of the first filtrate to 10.5 to 11.0 with 20% (w/v) sodium hydroxide solution, add 6 ⁇ l of hydrogen peroxide, and stir the reaction for 8 hours.
  • the first purified liquid was obtained.
  • the first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 5000 Da, and subjected to ultrafiltration for 10 hours, and 180 ⁇ l of the first retentate was collected.
  • Alcohol precipitation 144 ⁇ l of methanol was added to the first retentate obtained in the step 3), and the precipitate was precipitated for 18 hours to obtain a primary precipitate; the primary precipitate was dissolved in water to 180 ⁇ l, and 216 ⁇ l of methanol was added to precipitate for 18 hours. The first precipitate was isolated and the first precipitate was dried in vacuo and weighed (37 mg).
  • step 5 Degradation: add 296 mg of water to the first precipitate obtained in step 4), adjust the pH to 3.0-4.0 with 2 mol/L hydrochloric acid solution, adjust the temperature to 30 ° C, add 1.11 mg of sodium nitrite, and stir the reaction for 120 minutes. , the first liquid is obtained.
  • the second filtrate was filtered, and 5 volumes of pharmaceutically acceptable ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 15 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 300 ⁇ l, stirred to dissolve, and ultraviolet irradiation was performed for 30 minutes to obtain a fourth liquid.
  • the second purification liquid obtained in the step 7) is subjected to ultrafiltration through a membrane of 6000 Da, and the second retentate is collected: the second retentate is ultrafiltered through a membrane of 0.22 ⁇ m to obtain a third filtrate, and the third adjustment is performed.
  • the filtrate has a pH of 7 to 7.4.
  • Enzymatic hydrolysis Weigh 40 mg of crude heparin sodium, add 500 mg of water to it, dissolve the raw material liquid; adjust the raw materials The temperature of the solution was adjusted to 36 ° C, and 0.43 mg of papain, 0.32 mg of ribonuclease II and 0.32 mg of deoxyribonuclease I were respectively added, and the reaction was stirred for 13 hours, and the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
  • control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 10.0 to 10.5 with 20% (w/v) sodium hydroxide solution, add 5 ⁇ l of t-butyl hydroperoxide, and stir. The reaction was carried out for 8 hours to obtain a first purified liquid.
  • the first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cutoff of 4000 Da, and subjected to ultrafiltration for 8 hours, and 160 ⁇ l of the first retentate was collected.
  • Alcohol precipitation Add 130 ⁇ l of methanol to the first retentate obtained in step 3), precipitate for 16 hours, and separate the primary precipitate; then dissolve the primary precipitate into water to 160 ⁇ l, add 180 ⁇ l of methanol, and precipitate for 17 hours. The first precipitate was isolated and the first precipitate was dried in vacuo and weighed (36.4 mg).
  • step 5 Degradation: add 280 mg of water to the first precipitate obtained in step 4), adjust the pH to 3.0-3.5 with 2 mol/L hydrochloric acid solution, adjust the temperature to 25 ° C, add 0.78 mg of sodium nitrite, and stir the reaction for 100 minutes. , the first liquid is obtained.
  • the second filtrate was filtered, and 4 volumes of medicinal ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 13 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 260 ⁇ l, stirred to dissolve, and ultraviolet irradiation was carried out for 30 minutes to obtain a fourth liquid.
  • the second purification liquid obtained in the step 7) is subjected to ultrafiltration through a 5000 Da membrane, and the second retentate is collected: the second retentate is ultrafiltered through a 0.25 ⁇ m membrane to obtain a third filtrate, and the third adjustment is performed.
  • the filtrate has a pH of 7 to 7.4.
  • Enzymatic hydrolysis 40 mg of crude heparin sodium was weighed, 460 mg of water was added thereto, and the raw material liquid was dissolved; the temperature of the raw material liquid was adjusted to 36 ° C, and 0.53 mg of papain, 0.32 mg of ribonuclease II, and deoxyribonuclease I were respectively added. 0.2 mg, the reaction was stirred for 12 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
  • control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, add 8 mg of sodium peroxide, and stir the reaction for 8 hours.
  • the first purified liquid was obtained.
  • the first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 3000 Da, and subjected to ultrafiltration for 10 hours, and 180 ⁇ l of the first retentate was collected.
  • Alcohol precipitation Add 140 ⁇ l of methanol to the first retentate obtained in step 3), precipitate for 15 hours, and separate the primary precipitate; then dissolve the primary precipitate into water to 180 ⁇ l, add 180 ⁇ l of methanol, and precipitate for 16 hours. The first precipitate was isolated, and the first precipitate was vacuum dried and weighed (35.6 mg).
  • step 5 Degradation: 260 mg of water was added to the first precipitate obtained in step 4), the pH was adjusted to 2.0 to 2.5 with 2 mol/L hydrochloric acid solution, the temperature was adjusted to 20 ° C, 0.76 mg of sodium nitrite was added, and the reaction was stirred for 90 minutes. , the first liquid is obtained.
  • the second filtrate was filtered, and 4 volumes of medicinal ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 13 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 260 ⁇ l, stirred to dissolve, and ultraviolet irradiation was carried out for 30 minutes to obtain a fourth liquid.
  • the second purified liquid obtained in the step 7) is subjected to ultrafiltration through a 4000 Da membrane, and the second retentate is collected to obtain a second retentate.
  • the second retentate is ultrafiltered through a membrane of 0.2 ⁇ m to obtain a third filtrate, and the third filtrate is adjusted.
  • the filtrate pH was between 6.5 and 7.0.
  • Test example Activity detection of samples by reference to the United States Pharmacopoeia method
  • the average molecular weight of the low molecular weight heparin sodium heparin standard library prepared by the method of the invention is 5800-6200, the molecular weight distribution: ⁇ 1600 ratio ⁇ 40.0%; > 4500 ratio ⁇ 11.0%; anti-FXa/FIIa> 30, anti-FXa titer: 195 ⁇ 210IU / mg, anti-FIIa titer: ⁇ 5.0IU / mg, compared with other low molecular weight heparin sodium, has very good stability and antithrombotic activity, product quality beyond the European Pharmacopoeia standards It is expected to become a new generation of heparin antithrombotic drugs.
  • the low molecular weight heparin sodium heparin standard library provided by the invention has a molecular weight of 5800 Da to 6200 Da, a molecular weight distribution: ⁇ 1600 ratio ⁇ 40.0%; > 4500 ratio ⁇ 11.0%. It has the advantages of high purity, concentrated molecular weight distribution, and meets the stringent requirements for standard products in scientific research activities. It is often used as a standard in scientific research activities.

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Abstract

The present invention provides a method for preparing a standard library of a low-molecular-weight heparin, dalteparin sodium, and a product thereof. The method uses crude heparin sodium as a raw material and is based on compound enzymatic hydrolysis and a modified nitrite degradation method. After the steps of enzymatic hydrolysis, oxidation, purification by ultrafiltration, purification by ethanol precipitation, degradation, reduction, alkaline oxidation and purification, refining by ultrafiltration, freeze drying, etc., a standard library of a low-molecular-weight heparin, dalteparin sodium, with a particular average molecular weight can be prepared.

Description

一种低分子肝素达肝素钠标准品库及其制备方法Low molecular weight heparin heparin sodium standard product library and preparation method thereof 技术领域Technical field
本发明涉及生物医药领域，具体地说是一种低分子肝素达肝素钠标准品库及其制备方法。The invention relates to the field of biomedicine, in particular to a low molecular weight heparin sodium heparin standard library and a preparation method thereof.
背景技术Background technique
肝素(普通肝素即未分级肝素)是一种高度硫酸化的糖胺聚糖，在体内和体外都有抗凝血作用。但普通肝素存在生物利用度低、副作用大和给药次数过频等缺陷，而其换代产品低分子肝素如达特安瑞不仅抗血栓作用优于普通肝素，而且具有生物利用度高、体内半衰期长、出血倾向小等优点。肝素的抗凝血活性分为抗栓活性(FXa)和抗凝(FIIa)活性两大类，在抗血栓形成的过程中，抗FXa活性与抗FIIa活性的作用都是必需的，抗血栓作用的衡量标准用抗FXa/FIIa值表示，其值越大，表示抗血栓作用越强，出血倾向越小。低分子达特安瑞是由经亚硝酸降解而得到的低分子肝素的一种，是普通肝素的一种升级产品，相对平均分子量在5600～6400Da之间，平均值在6000Da，其主要组成部分为在糖链的非还原末端具有2位氧硫酸化的艾多糖醛酸，在糖链的还原末端具有6位氧硫酸化的2,5-脱氢甘露醛。用于治疗急性深静脉血栓；急性肾功能衰竭和慢性肾功能不全者进行血液透析和血液过滤期间防止在体外循环***中发生凝血；不稳定型冠状动脉疾病；预防与手术有关的血栓形成。具有体内半衰期延长、生物利用度高等优点，是目前主流的抗血栓类的药物。Heparin (unfractionated heparin, unfractionated heparin) is a highly sulfated glycosaminoglycan that has anticoagulant effects both in vivo and in vitro. However, unfractionated heparin has defects such as low bioavailability, large side effects, and excessive frequency of administration, and its replacement product, low molecular weight heparin such as Dartare is not only superior to unfractionated heparin, but also has high bioavailability and long half-life in vivo. , bleeding tendency is small and so on. The anticoagulant activity of heparin is divided into two major categories: antithrombotic activity (FXa) and anticoagulant (FIIa) activity. In the antithrombotic process, anti-FXa activity and anti-FIIa activity are essential, antithrombotic effect. The measure is expressed by the anti-FXa/FIIa value, and the larger the value, the stronger the antithrombotic effect and the smaller the bleeding tendency. Low molecular Dartare is a kind of low molecular weight heparin obtained by nitrite degradation. It is an upgraded product of unfractionated heparin. The relative average molecular weight is between 5600 and 6400 Da, and the average value is 6000 Da. In order to have an oxygenated sulfated albinoic acid at the non-reducing end of the sugar chain, there is a 6-oxygenated 2,5-dehydromannuraldehyde at the reducing end of the sugar chain. For the treatment of acute deep venous thrombosis; acute renal failure and chronic renal insufficiency during hemodialysis and hemofiltration to prevent coagulation in the extracorporeal circulation system; unstable coronary artery disease; prevention of surgery-related thrombosis. It has the advantages of prolonged half-life in vivo and high bioavailability, and is currently the mainstream antithrombotic drug.
低分子达特安瑞(Dalteparin Sodium)是未分级肝素在特定的温度和pH条件下，与亚硝酸盐作用，使未分级肝素中的N-硫酸葡萄糖胺反应脱去HSO4-形成-NH2，-NH2与亚硝酸盐发生重氮化反应，在放出N2的同时糖苷键断裂，电子转移，缩环生成2,5-脱氢甘露醛，后者经还原糖或脱氢形成甘露糖醇。它与肝素产品相比，有着更好的抗血栓活性，更小的出血危险，以及更低的对血小板的影响。但是在现有技术中，制备的低分子肝素样品拥有纯度不够、分子量分布不集中、杂质残留过多等缺点。而想要利用低分子肝素达肝素钠形成可以用于研究的标准品库，则需要高品质、高质量的低分子肝素达肝素钠，这些都是目前的工艺所不能解决的问题。Low-molecular Dalteparin Sodium is an unfractionated heparin that reacts with nitrite at specific temperature and pH conditions to react N-glucosamine sulfate in unfractionated heparin to remove HSO 4 - form -NH 2 , -NH 2 undergoes diazotization reaction with nitrite, cleavage of glycosidic bond while releasing N 2 , electron transfer, condensed ring to form 2,5-dehydromannuraldehyde, and the latter undergoes reducing sugar or dehydrogenation to form mannose alcohol. It has better antithrombotic activity, less bleeding risk, and lower platelet impact than heparin products. However, in the prior art, the prepared low molecular weight heparin sample has disadvantages such as insufficient purity, inconsistent molecular weight distribution, and excessive residual impurities. The use of low molecular weight heparin sodium heparin to form a standard library that can be used for research requires high quality, high quality low molecular weight heparin sodium, which is a problem that cannot be solved by current processes.
发明内容Summary of the invention
本发明的第一目的是针对上述现有技术的不足，提供一种低分子肝素达肝素钠标准品 库的制备方法。与其他低分子肝素钠相比，该方法制备的高品质低分子肝素达肝素钠标准品库有着非常好的稳定性和抗血栓活性。A first object of the present invention is to provide a low molecular weight heparin sodium heparin standard for the above-mentioned deficiencies of the prior art. The preparation method of the library. Compared with other low molecular weight heparin sodium, the high quality low molecular weight heparin sodium heparin standard library prepared by this method has very good stability and antithrombotic activity.
本发明的第二目的在于提供一种低分子肝素达肝素钠标准品库，其由上述低分子肝素达肝素钠标准品库的制备方法制备得到，具有纯度高、分子量分布集中、抗血栓活性好的优点。A second object of the present invention is to provide a low molecular weight heparin sodium heparin standard library prepared by the preparation method of the above low molecular weight heparin sodium heparin standard library, which has high purity, concentrated molecular weight distribution and good antithrombotic activity. The advantages.
本发明的技术任务是按以下方式实现的：The technical task of the present invention is achieved in the following manner:
一种低分子肝素达肝素钠标准品库的制备方法，包括以下步骤：A method for preparing a low molecular weight heparin sodium heparin standard library comprises the following steps:
1)酶解：向粗品肝素钠中加入10～15倍量水(质量比)，溶解得原料液，调节原料液温度至35℃～39℃，加入复合酶，搅拌反应10～15小时，去沉淀，上清液过滤，收集滤液；1) Enzymatic hydrolysis: Add 10 to 15 times the amount of water (mass ratio) to the crude heparin sodium, dissolve the raw material solution, adjust the temperature of the raw material liquid to 35 ° C to 39 ° C, add the complex enzyme, and stir the reaction for 10 to 15 hours. Precipitate, filter the supernatant, and collect the filtrate;
2)氧化：控制步骤1)所得滤液温度至40℃～50℃，调整滤液pH值至9～11，加入0.1％～0.5％滤液体积的过氧化氢，搅拌反应6～8小时，得纯化液；2) Oxidation: control the temperature of the filtrate obtained in step 1) to 40 ° C ~ 50 ° C, adjust the pH of the filtrate to 9 ~ 11, add 0.1% ~ 0.5% filtrate volume of hydrogen peroxide, stir the reaction for 6-8 hours, to obtain a purification solution ;
3)超滤除杂：采用截留分子量为3,000Da～5,000Da的超滤膜对步骤2)所得纯化液进行切向流循环超滤，循环超滤5～10小时，收集截留液；3) ultrafiltration and impurity removal: using the ultrafiltration membrane with molecular weight cut off of 3,000 Da to 5,000 Da, the purified liquid obtained in step 2) is subjected to tangential flow circulation ultrafiltration, and circulating ultrafiltration for 5 to 10 hours to collect the retentate;
4)醇沉除杂：截留液醇沉除杂，沉淀物干燥称重；4) Alcohol precipitation and impurity removal: the retentate alcohol is removed and the precipitate is dried and weighed;
5)降解：向步骤4)所得沉淀物中加7～8倍量的水溶解，用盐酸溶液调节料液pH值至2.0～4.0，调整料液温度至15～30℃，加入适量亚硝酸钠，搅拌反应90～120分钟；所述亚硝酸钠的加入量为步骤4)所得沉淀物质量的2％～3％；5) Degradation: 7-8 times of water is added to the precipitate obtained in step 4), the pH of the solution is adjusted to 2.0-4.0 with hydrochloric acid solution, the temperature of the solution is adjusted to 15-30 ° C, and an appropriate amount of sodium nitrite is added. Agitating the reaction for 90 to 120 minutes; the sodium nitrite is added in an amount of from 2% to 3% by mass of the precipitate obtained in the step 4);
6)还原：降解反应完成后，迅速用氢氧化钠溶液调节料液的pH值至9.0～10.0，加入适量硼氢化钠，搅拌反应10～15小时，反应完成后用盐酸溶液调节pH值至2.0～5.0，继续搅拌反应15～30分钟，反应完成后，用氢氧化钠溶液调节料液的pH值至6～8，并加入适量的氯化钠，搅拌溶解并过滤，向滤液中加入3～5倍体积的醇液，搅拌后静置12～15小时；去上清液，向沉淀物中加入水，搅拌使其溶解，并进行20～30分钟紫外照射；所述硼氢化钠的加入量为步骤4)所述沉淀物质量的0.9％～1.1％；氯化钠与料液的质量体积比为0.9％～1.1％；6) Reduction: After the completion of the degradation reaction, quickly adjust the pH of the solution to 9.0 to 10.0 with sodium hydroxide solution, add appropriate amount of sodium borohydride, and stir the reaction for 10 to 15 hours. After the reaction is completed, adjust the pH to 2.0 with hydrochloric acid solution. ～5.0, continue to stir the reaction for 15 to 30 minutes, after the reaction is completed, adjust the pH of the feed solution to 6-8 with sodium hydroxide solution, add appropriate amount of sodium chloride, stir to dissolve and filter, add 3~ to the filtrate 5 times the volume of the alcohol solution, after stirring, let stand for 12 to 15 hours; remove the supernatant, add water to the precipitate, stir to dissolve, and perform UV irradiation for 20 to 30 minutes; the amount of the sodium borohydride added The step (1) is 0.9% to 1.1% of the mass of the precipitate; the mass to volume ratio of sodium chloride to the feed liquid is 0.9% to 1.1%;
7)碱氧纯化：用氢氧化钠溶液调节料液pH值至9～10，加入1～3％料液体积的过氧化氢，20～30℃料液温度下搅拌反应6～8小时；氧化完毕后，醇沉，并将沉淀物用适量水溶解，所用水的量为步骤4)所述沉淀物质量的3～4倍；7) Purification of alkali oxygen: adjust the pH of the solution to 9-10 with sodium hydroxide solution, add 1 to 3% of the volume of hydrogen peroxide, and stir the reaction at 20 to 30 ° C for 6-8 hours; oxidation After completion, alcohol precipitation, and the precipitate is dissolved with an appropriate amount of water, the amount of water used is 3 to 4 times the mass of the precipitate in step 4);
8)超滤精制：将步骤7)所得溶液用5000Da～6000Da的膜循环超滤，收集截留液；截留液经0.22μm的膜超滤，得滤液，调节滤液pH值至6.4～7.4；8) ultrafiltration purification: the solution obtained in step 7) is ultrafiltered with a membrane of 5000 Da to 6000 Da, and the retentate is collected; the retentate is ultrafiltered through a membrane of 0.22 μm to obtain a filtrate, and the pH of the filtrate is adjusted to 6.4 to 7.4;
9)冻干：将滤液冻干，得低分子肝素达肝素钠标准品库。9) Lyophilization: The filtrate was lyophilized to obtain a low molecular weight heparin to a standard library of heparin sodium.
优选的，所述复合酶包括木瓜蛋白酶、核糖核酸酶II及脱氧核糖核酸酶I，木瓜蛋白酶的用量为肝素钠粗品重量的1％～2％，核糖核酸酶II的用量为肝素钠粗品重量的0.5％～1％， 脱氧核糖核酸酶I的用量为肝素钠粗品重量的0.5％～1％。Preferably, the complex enzyme comprises papain, ribonuclease II and deoxyribonuclease I, the amount of papain is 1% to 2% of the weight of the crude heparin sodium, and the amount of ribonuclease II is the weight of the crude heparin sodium. 0.5% to 1%, The amount of DNase I is from 0.5% to 1% by weight of the crude heparin sodium.
步骤4)、步骤7)中，可以利用现有技术中公知的醇沉方法完成醇沉操作，但是，为了得到更好的提纯效果，优选以下具体方法实现：In step 4) and step 7), the alcohol precipitation operation can be carried out by the alcohol precipitation method well known in the prior art, but in order to obtain a better purification effect, the following specific methods are preferably implemented:
步骤4)醇沉除杂时，向步骤3)所得的截留液中加入0.6～0.8倍体积的甲醇，沉淀14～18小时，分离得沉淀物；再将沉淀物加水溶解，向溶液中加入1～1.2倍体积的甲醇，沉淀14～18小时，分离得沉淀物，将沉淀物真空干燥并称重；Step 4) When the alcohol is removed, the 0.6 to 0.8 volume of methanol is added to the retentate obtained in the step 3), and the precipitate is precipitated for 14 to 18 hours to separate the precipitate; the precipitate is dissolved in water, and 1 is added to the solution. ~ 1.2 times the volume of methanol, precipitated for 14 to 18 hours, the precipitate was separated, and the precipitate was vacuum dried and weighed;
步骤7)醇沉的具体过程：用盐酸溶液调节料液pH至6～8，向其中加入适量氯化钠及2～3倍体积的乙醇，搅拌20～30分钟以后，静置沉淀8～10小时，去上清液；氯化钠与料液的质量体积比为1％～3％。Step 7) The specific process of alcohol precipitation: adjust the pH of the feed solution to 6-8 with hydrochloric acid solution, add appropriate amount of sodium chloride and 2 to 3 volumes of ethanol, stir for 20 to 30 minutes, and then settle the precipitate 8 to 10 In the hour, the supernatant is removed; the mass to volume ratio of sodium chloride to the feed liquid is 1% to 3%.
步骤6)中，所述醇液优选为甲醇溶液或乙醇溶液。In the step 6), the alcohol liquid is preferably a methanol solution or an ethanol solution.
一种低分子肝素达肝素钠标准品库，由上述低分子肝素达肝素钠标准品库的制备方法制备得到，低分子肝素达肝素钠标准品库的分子量为5800Da～6200Da，分子量分布：＜1600的比例≤40.0％；＞4500的比例≤11.0％。A low molecular weight heparin sodium heparin standard library is prepared by the preparation method of the low molecular weight heparin heparin sodium standard library. The molecular weight of the low molecular weight heparin sodium heparin standard library is 5800Da to 6200Da, and the molecular weight distribution is <1600. The ratio is ≤ 40.0%; the ratio of > 4500 is ≤ 11.0%.
与现有技术相比，本发明的低分子肝素达肝素钠标准品库的制备方法具有以下突出的Compared with the prior art, the preparation method of the low molecular weight heparin sodium heparin standard library of the invention has the following outstanding
有益效果：Beneficial effects:
(一)以复合酶解和改良的亚硝酸盐降解法为基础，以粗品肝素钠为原料，选用木瓜蛋白酶、核糖核酸酶II等蛋白和核酸酶优化组合，对粗品肝素中蛋白和核酸进行高效降解，并结合膜技术一次性去除蛋白和核酸杂质，避免了传统工艺中从粗品到精品制备反复除杂的繁琐过程；(1) Based on the composite enzymatic hydrolysis and modified nitrite degradation method, using crude heparin sodium as raw material, using papain, ribonuclease II and other proteins and nucleases to optimize the combination of proteins and nucleic acids in crude heparin Degradation, combined with membrane technology to remove protein and nucleic acid impurities at one time, avoiding the cumbersome process of repeated decontamination from crude to fine preparation in traditional processes;
(二)未分级肝素在特定的温度和pH条件下，与亚硝酸盐作用，使未分级肝素中的N-硫酸葡萄糖胺反应脱去HSO4-形成-NH2，-NH2与亚硝酸盐发生重氮化反应，在放出N2的同时糖苷键断裂，电子转移，缩环生成2,5-脱氢甘露醛，后者经还原糖或脱氢形成甘露糖醇，后通过低分子膜分离技术获得具有特定平均分子量(5800-6200)的低分子肝素达肝素钠标准品库；(2) Unfractionated heparin reacts with nitrite at a specific temperature and pH to desorb HSO 4 in the unfractionated heparin to form HSO 4 - form -NH 2 , -NH 2 and nitrite The diazotization reaction occurs, the glycosidic bond is broken while the N 2 is released, and the electron transfer and condensed ring form 2,5-dehydromannuraldehyde, which is formed by reducing sugar or dehydrogenation to form mannitol, and then separated by a low molecular membrane. The technology obtains a library of low molecular weight heparin sodium heparin standards with a specific average molecular weight (5800-6200);
(三)通过本发明方法制备得到的低分子肝素达肝素钠标准品库平均分子量为5800～6200，分子量分布：＜1600的比例≤40.0％；＞4500的比例≤11.0％；抗FXa/FIIa＞30，抗FXa效价：195～210IU/mg，抗FIIa效价：＜5.0IU/mg，与其他低分子肝素钠相比，有着非常好的稳定性和抗血栓活性，产品质量超越欧洲药典标准，有望成为新生代的肝素类抗血栓药物。(3) The average molecular weight of the low molecular weight heparin sodium heparin standard library prepared by the method of the invention is 5800-6200, the molecular weight distribution: <1600 ratio ≤ 40.0%; > 4500 ratio ≤ 11.0%; anti-FXa/FIIa> 30, anti-FXa titer: 195 ~ 210IU / mg, anti-FIIa titer: <5.0IU / mg, compared with other low molecular weight heparin sodium, has very good stability and antithrombotic activity, product quality beyond the European Pharmacopoeia standards It is expected to become a new generation of heparin antithrombotic drugs.
(四)本发明所提供的低分子肝素达肝素钠标准品库，其分子量为5800Da～6200Da，分子量分布：＜1600的比例≤40.0％；＞4500的比例≤11.0％。具有纯度高、分子量分布集中等优点，达到科研活动中的对于标准品的严苛要求，非常适合作为科研活动中的标准品 使用。(4) The low molecular weight heparin sodium heparin standard library provided by the invention has a molecular weight of 5800 Da to 6200 Da, a molecular weight distribution: <1600 ratio ≤ 40.0%; > 4500 ratio ≤ 11.0%. It has the advantages of high purity, concentrated molecular weight distribution, and meets the stringent requirements for standard products in scientific research activities. It is very suitable as a standard in scientific research activities. use.
具体实施方式Detailed ways
以具体实施例对本发明的低分子肝素达肝素钠标准品库的制备方法作以下详细地说明。The preparation method of the low molecular weight heparin sodium heparin standard library of the present invention will be described in detail below with reference to specific examples.
制备实施例1：Preparation Example 1:
具体步骤：Specific steps:
1)酶解：称取粗品肝素钠20mg，向其中加入240mg水，溶解得原料液；调节原料液温度至37℃，分别加入木瓜蛋白酶0.2mg、核糖核酸酶II 0.1mg及脱氧核糖核酸酶I 0.1mg，搅拌反应12小时，去沉淀，上清液过滤，收集得第一滤液。1) Enzymatic hydrolysis: Weigh 20 mg of crude heparin sodium, add 240 mg of water to it, dissolve the raw material solution; adjust the temperature of the raw material solution to 37 ° C, add 0.2 mg of papain, 0.1 mg of ribonuclease II and deoxyribonuclease I, respectively. 0.1 mg, the reaction was stirred for 12 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
2)氧化：控制步骤1)所得第一滤液温度至45℃，用20％(w/v)氢氧化钠溶液调整第一滤液的pH值至9.5～10，加入2.4μl过氧化氢，搅拌反应7小时，得第一纯化液。2) Oxidation: control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 9.5-10 with 20% (w/v) sodium hydroxide solution, add 2.4 μl of hydrogen peroxide, and stir the reaction. After 7 hours, the first purified liquid was obtained.
3)超滤除杂：采用截留分子量为4,000Da的超滤膜对步骤2)所得第一纯化液进行切向流循环超滤，循环超滤7小时，收集得第一截留液63.2μl。3) Ultrafiltration removal: The first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 4,000 Da, and subjected to ultrafiltration for 7 hours to collect 63.2 μl of the first retentate.
4)醇沉除杂：向步骤3)所得的第一截留液中加入50.4μl甲醇，沉淀16小时，分离得初级沉淀物；再将初级沉淀物加水溶解至60μl，并加入60μl甲醇，沉淀16小时，分离得第一沉淀物，将第一沉淀物真空干燥并称重(16.6mg)。4) Alcohol precipitation: 50.4 μl of methanol was added to the first retentate obtained in the step 3), and the precipitate was precipitated for 16 hours to obtain a primary precipitate; the primary precipitate was dissolved in water to 60 μl, and 60 μl of methanol was added to precipitate 16 After the hour, the first precipitate was separated, and the first precipitate was vacuum dried and weighed (16.6 mg).
5)降解：向步骤4)所得第一沉淀物中加120mg水溶解，用2mol/L盐酸溶液调节pH值至2.0～4.0，调整温度至15℃，加入0.4mg亚硝酸钠，搅拌反应90分钟，得到第一料液。5) Degradation: 120 mg of water was added to the first precipitate obtained in step 4), the pH was adjusted to 2.0 to 4.0 with 2 mol/L hydrochloric acid solution, the temperature was adjusted to 15 ° C, 0.4 mg of sodium nitrite was added, and the reaction was stirred for 90 minutes. , the first liquid is obtained.
6)还原：降解反应完成后，迅速用20％(w/v)的氢氧化钠溶液调节第一料液的pH值至9.0～10.0，加入0.17mg硼氢化钠，搅拌反应12小时，得到第二料液。反应完成后用4mol/L的盐酸溶液调节第二料液的pH值至2.0～5.0，继续搅拌反应20分钟，得到第三料液。反应完成后，再用20％(w/v)氢氧化钠溶液调节第三料液的pH值至6.5～7，并加入1％(w/v)第三料液体积的NaCl，搅拌溶解并过滤，得到第二滤液，向第二滤液中加入4倍体积的药用乙醇，搅拌后静置14小时，去上清液，得到第二沉淀物。向第二沉淀物中加入水至120μl，搅拌使其溶解，并进行25分钟紫外照射，得到第四料液。6) Reduction: After the degradation reaction is completed, the pH of the first liquid is adjusted to 9.0 to 10.0 with 20% (w/v) sodium hydroxide solution, 0.17 mg of sodium borohydride is added, and the reaction is stirred for 12 hours to obtain the first Two liquids. After the completion of the reaction, the pH of the second liquid was adjusted to 2.0 to 5.0 with a 4 mol/L hydrochloric acid solution, and the reaction was further stirred for 20 minutes to obtain a third liquid. After the reaction is completed, the pH of the third liquid is adjusted to 6.5-7 with 20% (w/v) sodium hydroxide solution, and 1% (w/v) of the third liquid volume of NaCl is added, stirred and dissolved. After filtration, a second filtrate was obtained, and 4 volumes of pharmaceutically acceptable ethanol was added to the second filtrate, and after stirring, it was allowed to stand for 14 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 120 μl, stirred to dissolve, and ultraviolet irradiation was carried out for 25 minutes to obtain a fourth liquid.
7)碱氧纯化：用20％(w/v)的氢氧化钠溶液调节第四料液pH值至9.5～10，加入1.5％第四料液体积的过氧化氢，25℃的温度下搅拌反应7小时，得到第五料液。氧化完毕后，用lmol/L的盐酸溶液调节第五料液的pH至6.5～7，向其中加入1.5％(w/v)第五料液体积的NaCl及2.5倍第五料液体积的乙醇，搅拌25分钟以后，静置沉淀9小时，去上清液，得到第三沉淀物。将第三沉淀物用60mg水溶解，得到第二纯化液。 7) Alkaline oxygen purification: adjust the pH of the fourth liquid to 9.5-10 with 20% (w/v) sodium hydroxide solution, add 1.5% of the fourth liquid volume of hydrogen peroxide, and stir at 25 °C. The reaction was carried out for 7 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth liquid solution is adjusted to 6.5-7 with a 1 mol/L hydrochloric acid solution, and 1.5% (w/v) of the fifth liquid volume of NaCl and 2.5 times of the fifth liquid volume of ethanol are added thereto. After stirring for 25 minutes, the precipitate was allowed to stand for 9 hours, and the supernatant was removed to obtain a third precipitate. The third precipitate was dissolved in 60 mg of water to obtain a second purified solution.
8)超滤精制：将步骤7)所得第二纯化液用5000Da的膜循环超滤，收集得到第二截留液。第二截留液经0.22μm的膜超滤，得第三滤液，调节第三滤液的pH值至6.4～7。8) Ultrafiltration purification: The second purified liquid obtained in the step 7) was subjected to ultrafiltration through a 5000 Da membrane, and the second retentate was collected. The second retentate was ultrafiltered through a 0.22 μm membrane to obtain a third filtrate, and the pH of the third filtrate was adjusted to 6.4-7.
9)冻干：将将调节pH后第三滤液冻干，得低分子肝素达肝素钠标准品库成品8.6mg。9) Lyophilization: The third filtrate will be lyophilized after adjusting the pH to obtain 8.6 mg of low molecular weight heparin and heparin sodium standard library.
制备实施例2：Preparation Example 2:
具体步骤：Specific steps:
1)酶解：称取粗品肝素钠40mg，向其中加入400mg水，溶解得原料液；调节原料液温度至37℃，分别加入木瓜蛋白酶0.8mg、核糖核酸酶II 0.2mg及脱氧核糖核酸酶I0.2mg，搅拌反应12小时，去沉淀，上清液过滤，收集得第一滤液。1) Enzymatic hydrolysis: Weigh 40 mg of crude heparin sodium, add 400 mg of water to it, dissolve the raw material solution; adjust the temperature of the raw material solution to 37 ° C, and add papain 0.8 mg, ribonuclease II 0.2 mg and deoxyribonuclease I0, respectively. .2 mg, the reaction was stirred for 12 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
2)氧化：控制步骤1)所得第一滤液温度至45℃，用20％(w/v)氢氧化钠溶液调整第一滤液的pH值至9.5～10，加入5μl过氧化氢，搅拌反应7小时，得第一纯化液。2) Oxidation: control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 9.5-10 with 20% (w/v) sodium hydroxide solution, add 5 μl of hydrogen peroxide, and stir the reaction 7 In hours, the first purified liquid was obtained.
3)超滤除杂：采用截留分子量为4000Da的超滤膜对步骤2)所得第一纯化液进行切向流循环超滤，循环超滤7小时，收集得第一截留液140μl。3) Ultrafiltration removal: The first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 4000 Da, and subjected to ultrafiltration for 7 hours, and 140 μl of the first retentate was collected.
4)醇沉除杂：向步骤3)所得的第一截留液中加入112μl甲醇，沉淀16小时，分离得初级沉淀物。再将初级沉淀物加水溶解至140μl，并加入160μl甲醇，沉淀16小时，分离得第一沉淀物，将第一沉淀物真空干燥并称重(34.4mg)。4) Alcohol precipitation: To the first retentate obtained in the step 3), 112 μl of methanol was added, and the precipitate was precipitated for 16 hours to obtain a primary precipitate. The primary precipitate was further dissolved in water to 140 μl, and 160 μl of methanol was added thereto, and precipitated for 16 hours, and the first precipitate was separated, and the first precipitate was vacuum dried and weighed (34.4 mg).
5)降解：向步骤4)所得第一沉淀物中加240mg水溶解，用2mol/L盐酸溶液调节pH值至2.0～4.0，调整温度至30℃，加入0.8mg亚硝酸钠，搅拌反应120分钟，得到第一料液。5) Degradation: add 240 mg of water to the first precipitate obtained in step 4), adjust the pH to 2.0 to 4.0 with 2 mol/L hydrochloric acid solution, adjust the temperature to 30 ° C, add 0.8 mg of sodium nitrite, and stir the reaction for 120 minutes. , the first liquid is obtained.
6)还原：降解反应完成后，迅速用20％(w/v)的氢氧化钠溶液调节第一料液的pH值至9.0～10.0，加入0.34mg硼氢化钠，搅拌反应12小时，得到第二料液。反应完成后用4mol/L的盐酸溶液调节第二料液的pH值至2.0～5.0，继续搅拌反应30分钟，得到第三料液。反应完成后，再用20％(w/v)的氢氧化钠溶液调节第三料液的pH值至6.5～7，并加入1％(w/v)第三料液体积的NaCl，搅拌溶解并过滤，得到第二滤液。向第二滤液中加入4倍体积的药用乙醇，搅拌后静置14小时，去上清液，得到第二沉淀物。向第二沉淀物中加入水至240μl，搅拌使其溶解，并进行25分钟紫外照射，得到第四料液。6) Reduction: After the degradation reaction is completed, the pH of the first liquid is adjusted to 9.0 to 10.0 with 20% (w/v) sodium hydroxide solution, 0.34 mg of sodium borohydride is added, and the reaction is stirred for 12 hours to obtain the first Two liquids. After the completion of the reaction, the pH of the second liquid was adjusted to 2.0 to 5.0 with a 4 mol/L hydrochloric acid solution, and the reaction was further stirred for 30 minutes to obtain a third liquid. After the reaction is completed, adjust the pH of the third liquid to 6.5-7 with 20% (w/v) sodium hydroxide solution, and add 1% (w/v) of the third liquid volume of NaCl, stir and dissolve. And filtering to obtain a second filtrate. To the second filtrate, 4 volumes of pharmaceutically acceptable ethanol was added, and after stirring, it was allowed to stand for 14 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 240 μl, stirred to dissolve, and ultraviolet irradiation was performed for 25 minutes to obtain a fourth liquid.
7)碱氧纯化：用20％(w/v)的氢氧化钠溶液调节第四料液的pH值至9.5～10，加入1.5％第四料液体积的过氧化氢，25℃料液温度下搅拌反应7小时，得到第五料液。氧化完毕后，用1mol/L的盐酸溶液调节第五料液的pH至6.5～7，向其中加入1.5％(w/v)第五料液体积的NaCl及2.5倍第五料液体积的乙醇，搅拌25分钟以后，静置沉淀9小时，去上清液，得到第三沉淀物。将第三沉淀物用120mg水溶解，得到第二纯化液。7) Alkaline oxygen purification: adjust the pH of the fourth liquid to 9.5-10 with 20% (w/v) sodium hydroxide solution, add 1.5% of the fourth liquid volume of hydrogen peroxide, and the temperature of the 25 °C solution The reaction was stirred for 7 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth liquid solution is adjusted to 6.5-7 with a 1 mol/L hydrochloric acid solution, and 1.5% (w/v) of the fifth liquid volume of NaCl and 2.5 times of the fifth liquid volume of ethanol are added thereto. After stirring for 25 minutes, the precipitate was allowed to stand for 9 hours, and the supernatant was removed to obtain a third precipitate. The third precipitate was dissolved in 120 mg of water to obtain a second purified solution.
8)超滤精制：将步骤7)所得第二纯化液用5000Da的膜循环超滤，收集得到第二截留液。第二截留液经0.22μm的膜超滤，得第三滤液，调节第三滤液的pH值至6.4～7。 8) Ultrafiltration purification: The second purified liquid obtained in the step 7) was subjected to ultrafiltration through a 5000 Da membrane, and the second retentate was collected. The second retentate was ultrafiltered through a 0.22 μm membrane to obtain a third filtrate, and the pH of the third filtrate was adjusted to 6.4-7.
9)冻干：将将调节pH后第三滤液冻干，得低分子肝素达肝素钠标准品库18.4mg。9) Lyophilization: The third filtrate will be lyophilized after adjusting the pH to obtain 18.4 mg of low molecular weight heparin sodium heparin standard library.
实施例3：Example 3:
具体步骤：Specific steps:
1)酶解：称取粗品肝素钠40mg，向其中加入400mg水，溶解得原料液；调节原料液温度至35℃，分别加入木瓜蛋白酶0.4mg、核糖核酸酶II 0.2mg及脱氧核糖核酸酶I0.2mg，搅拌反应10小时，去沉淀，上清液过滤，收集得第一滤液。1) Enzymatic hydrolysis: 40 mg of crude heparin sodium was weighed, 400 mg of water was added thereto, and the raw material liquid was dissolved; the temperature of the raw material liquid was adjusted to 35 ° C, and papain 0.4 mg, ribonuclease II 0.2 mg, and deoxyribonuclease I0 were respectively added. .2 mg, the reaction was stirred for 10 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
2)氧化：控制步骤1)所得第一滤液温度至40℃，用20％(w/v)氢氧化钠溶液调整第一滤液pH值至9.0～10.0，加入4μl过氧化氢，搅拌反应6小时，得第一纯化液。2) Oxidation: control step 1) to obtain a temperature of the first filtrate to 40 ° C, adjust the pH of the first filtrate to 9.0 to 10.0 with 20% (w/v) sodium hydroxide solution, add 4 μl of hydrogen peroxide, and stir the reaction for 6 hours. The first purified liquid was obtained.
3)超滤除杂：采用截留分子量为3000Da的超滤膜对步骤2)所得第一纯化液进行切向流循环超滤，循环超滤5小时，收集得第一截留液140μl。3) Ultrafiltration removal: The first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 3000 Da, and subjected to ultrafiltration for 5 hours, and 140 μl of the first retentate was collected.
4)醇沉除杂：向步骤3)所得的第一截留液中加入84μl甲醇，沉淀14小时，分离得初级沉淀物；再将初级沉淀物加水溶解至140μl，并加入140μl甲醇，沉淀14小时，分离得第一沉淀物，将第一沉淀物真空干燥并称重(32mg)。4) Alcohol precipitation: 84 μl of methanol was added to the first retentate obtained in step 3), and the precipitate was precipitated for 14 hours to separate a primary precipitate; the primary precipitate was dissolved in water to 140 μl, and 140 μl of methanol was added to precipitate for 14 hours. The first precipitate was isolated and the first precipitate was dried in vacuo and weighed (32 mg).
5)降解：向步骤4)所得第一沉淀物中加224mg水溶解，用2mol/L盐酸溶液调节pH值至2.0～3.0，调整温度至15℃，加入0.64mg亚硝酸钠，搅拌反应90分钟，得到第一料液。5) Degradation: Add 224 mg of water to the first precipitate obtained in step 4), adjust the pH to 2.0-3.0 with 2 mol/L hydrochloric acid solution, adjust the temperature to 15 ° C, add 0.64 mg of sodium nitrite, and stir the reaction for 90 minutes. , the first liquid is obtained.
6)还原：降解反应完成后，迅速用20％(w/v)的氢氧化钠溶液调节第一料液的pH值至9.0～10.0，加入0.29mg硼氢化钠，搅拌反应10小时，得到第二料液。反应完成后用4mol/L的盐酸溶液调节第二料液的pH值至2.0～3.0，继续搅拌反应15分钟，得到第三料液。反应完成后，再用20％(w/v)的氢氧化钠溶液调节第三料液的pH值至6.0～6.5，并加入0.9％(w/v)第三料液体积的NaCl，搅拌溶解并过滤得到第二滤液，向第二滤液中加入3倍体积的药用乙醇，搅拌后静置12小时，去上清液，得到第二沉淀物。向第二沉淀物中加入水至240μl，搅拌使其溶解，并进行20分钟紫外照射，得到第四料液。6) Reduction: After the degradation reaction is completed, the pH of the first liquid is adjusted to 9.0 to 10.0 with 20% (w/v) sodium hydroxide solution, 0.29 mg of sodium borohydride is added, and the reaction is stirred for 10 hours to obtain the first Two liquids. After the completion of the reaction, the pH of the second liquid was adjusted to 2.0 to 3.0 with a 4 mol/L hydrochloric acid solution, and the reaction was further stirred for 15 minutes to obtain a third liquid. After the reaction is completed, the pH of the third liquid solution is adjusted to 6.0-6.5 with 20% (w/v) sodium hydroxide solution, and 0.9% (w/v) of the third liquid volume of NaCl is added, and stirred and dissolved. The second filtrate was filtered, and 3 volumes of medicinal ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 12 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 240 μl, stirred to dissolve, and ultraviolet irradiation was carried out for 20 minutes to obtain a fourth liquid.
7)碱氧纯化：用20％(w/v)的氢氧化钠溶液调节第四料液的pH值至9.0～9.5，加入1％第四料液体积的过氧化氢，20℃的温度下搅拌反应6小时，得到第五料液。氧化完毕后，用1mol/L的盐酸溶液调节第五料液pH至6.0～6.5，向其中加入1％(w/v)第五料液体积的NaCl及2倍第五料液体积的乙醇，搅拌20分钟以后，静置沉淀8小时，去上清液，得到第三沉淀物，将第三沉淀物用120mg水溶解，得到第二纯化液。7) Alkaline oxygen purification: adjust the pH of the fourth liquid to 9.0-9.5 with 20% (w/v) sodium hydroxide solution, and add 1% of the fourth liquid volume of hydrogen peroxide at a temperature of 20 °C. The reaction was stirred for 6 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth solution is adjusted to 6.0 to 6.5 with a 1 mol/L hydrochloric acid solution, and 1% (w/v) of the fifth solution volume of NaCl and 2 times of the fifth solution volume of ethanol are added thereto. After stirring for 20 minutes, the precipitate was allowed to stand for 8 hours, and the supernatant was removed to obtain a third precipitate, and the third precipitate was dissolved in 120 mg of water to obtain a second purified solution.
8)超滤精制：将步骤7)所得第二纯化液用5000Da的膜循环超滤，收集得到第二截留液：第二截留液经0.22μm的膜超滤，得第三滤液，调节第三滤液pH值至6.4～7。8) Ultrafiltration purification: the second purification liquid obtained in the step 7) is subjected to ultrafiltration through a membrane of 5000 Da, and the second retentate is collected: the second retentate is ultrafiltered through a membrane of 0.22 μm to obtain a third filtrate, and the third adjustment is performed. The pH of the filtrate was adjusted to 6.4-7.
9)冻干：将将调节pH后第三滤液冻干，得低分子肝素达肝素钠标准品库17mg。9) Lyophilization: The third filtrate will be lyophilized after adjusting the pH to obtain a low molecular weight heparin to a heparin sodium standard library of 17 mg.
实施例4： Example 4:
具体步骤：Specific steps:
1)酶解：称取粗品肝素钠40mg，向其中加入600mg水，溶解得原料液；调节原料液温度至39℃，分别加入木瓜蛋白酶0.8mg、核糖核酸酶II 0.4mg及脱氧核糖核酸酶I0.4mg，搅拌反应15小时，去沉淀，上清液过滤，收集得第一滤液。1) Enzymatic hydrolysis: Weigh 40 mg of crude heparin sodium, add 600 mg of water to it, dissolve the raw material solution; adjust the temperature of the raw material solution to 39 ° C, and add papain 0.8 mg, ribonuclease II 0.4 mg and deoxyribonuclease I0, respectively. .4 mg, the reaction was stirred for 15 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
2)氧化：控制步骤1)所得第一滤液温度至50℃，用20％(w/v)氢氧化钠溶液调整第一滤液pH值至10.5～11.0，加入6μl过氧化氢，搅拌反应8小时，得第一纯化液。2) Oxidation: control step 1) to obtain a temperature of the first filtrate to 50 ° C, adjust the pH of the first filtrate to 10.5 to 11.0 with 20% (w/v) sodium hydroxide solution, add 6 μl of hydrogen peroxide, and stir the reaction for 8 hours. The first purified liquid was obtained.
3)超滤除杂：采用截留分子量为5000Da的超滤膜对步骤2)所得第一纯化液进行切向流循环超滤，循环超滤10小时，收集得第一截留液180μl。3) Ultrafiltration removal: The first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 5000 Da, and subjected to ultrafiltration for 10 hours, and 180 μl of the first retentate was collected.
4)醇沉除杂：向步骤3)所得的第一截留液中加入144μl甲醇，沉淀18小时，分离得初级沉淀物；再将初级沉淀物加水溶解至180μl，并加入216μl甲醇，沉淀18小时，分离得第一沉淀物，将第一沉淀物真空干燥并称重(37mg)。4) Alcohol precipitation: 144 μl of methanol was added to the first retentate obtained in the step 3), and the precipitate was precipitated for 18 hours to obtain a primary precipitate; the primary precipitate was dissolved in water to 180 μl, and 216 μl of methanol was added to precipitate for 18 hours. The first precipitate was isolated and the first precipitate was dried in vacuo and weighed (37 mg).
5)降解：向步骤4)所得第一沉淀物中加296mg水溶解，用2mol/L盐酸溶液调节pH值至3.0～4.0，调整温度至30℃，加入1.11mg亚硝酸钠，搅拌反应120分钟，得到第一料液。5) Degradation: add 296 mg of water to the first precipitate obtained in step 4), adjust the pH to 3.0-4.0 with 2 mol/L hydrochloric acid solution, adjust the temperature to 30 ° C, add 1.11 mg of sodium nitrite, and stir the reaction for 120 minutes. , the first liquid is obtained.
6)还原：降解反应完成后，迅速用20％(w/v)的氢氧化钠溶液调节第一料液的pH值至9.5～10.0，加入0.41mg硼氢化钠，搅拌反应15小时，得到第二料液。反应完成后用4mol/L的盐酸溶液调节第二料液的pH值至4.0～5.0，继续搅拌反应30分钟，得到第三料液。反应完成后，再用20％(w/v)的氢氧化钠溶液调节第三料液的pH值至7.5～8.0，并加入1.1％(w/v)第三料液体积的NaCl，搅拌溶解并过滤得到第二滤液，向第二滤液中加入5倍体积的药用乙醇，搅拌后静置15小时，去上清液，得到第二沉淀物。向第二沉淀物中加入水至300μl，搅拌使其溶解，并进行30分钟紫外照射，得到第四料液。6) Reduction: After the degradation reaction is completed, the pH of the first liquid is adjusted to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, 0.41 mg of sodium borohydride is added, and the reaction is stirred for 15 hours to obtain the first Two liquids. After the completion of the reaction, the pH of the second liquid was adjusted to 4.0 to 5.0 with a 4 mol/L hydrochloric acid solution, and the reaction was further stirred for 30 minutes to obtain a third liquid. After the reaction is completed, the pH of the third liquid is adjusted to 7.5-8.0 with 20% (w/v) sodium hydroxide solution, and 1.1% (w/v) of the third liquid volume of NaCl is added and stirred to dissolve. The second filtrate was filtered, and 5 volumes of pharmaceutically acceptable ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 15 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 300 μl, stirred to dissolve, and ultraviolet irradiation was performed for 30 minutes to obtain a fourth liquid.
7)碱氧纯化：用20％(w/v)的氢氧化钠溶液调节第四料液的pH值至9.5～10.0，加入3％第四料液体积的过氧化氢，30℃的温度下搅拌反应8小时，得到第五料液。氧化完毕后，用1mol/L的盐酸溶液调节第五料液pH至7.5～8.0，向其中加入3％(w/v)第五料液体积的NaCl及3倍第五料液体积的乙醇，搅拌20分钟以后，静置沉淀10小时，去上清液，得到第三沉淀物，将第三沉淀物用160mg水溶解，得到第二纯化液。7) Alkaline oxygen purification: adjust the pH of the fourth liquid to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, add 3% of the fourth liquid volume of hydrogen peroxide, at a temperature of 30 ° C The reaction was stirred for 8 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth liquid solution is adjusted to 7.5-8.0 with a 1 mol/L hydrochloric acid solution, and 3% (w/v) of the fifth liquid volume of NaCl and 3 times of the fifth liquid volume of ethanol are added thereto. After stirring for 20 minutes, the precipitate was allowed to stand for 10 hours, and the supernatant was removed to obtain a third precipitate, and the third precipitate was dissolved in 160 mg of water to obtain a second purified solution.
8)超滤精制：将步骤7)所得第二纯化液用6000Da的膜循环超滤，收集得到第二截留液：第二截留液经0.22μm的膜超滤，得第三滤液，调节第三滤液pH值至7～7.4。8) Ultrafiltration purification: the second purification liquid obtained in the step 7) is subjected to ultrafiltration through a membrane of 6000 Da, and the second retentate is collected: the second retentate is ultrafiltered through a membrane of 0.22 μm to obtain a third filtrate, and the third adjustment is performed. The filtrate has a pH of 7 to 7.4.
9)冻干：将调节pH后的第三滤液冻干，得低分子肝素达肝素钠标准品库19mg。9) Lyophilization: The third filtrate after pH adjustment was lyophilized to obtain 19 mg of a low molecular weight heparin sodium heparin standard library.
实施例5：Example 5:
具体步骤：Specific steps:
1)酶解：称取粗品肝素钠40mg，向其中加入500mg水，溶解得原料液；调节原料 液温度至36℃，分别加入木瓜蛋白酶0.48mg、核糖核酸酶II 0.32mg及脱氧核糖核酸酶I 0.32mg，搅拌反应13小时，去沉淀，上清液过滤，收集得第一滤液。1) Enzymatic hydrolysis: Weigh 40 mg of crude heparin sodium, add 500 mg of water to it, dissolve the raw material liquid; adjust the raw materials The temperature of the solution was adjusted to 36 ° C, and 0.43 mg of papain, 0.32 mg of ribonuclease II and 0.32 mg of deoxyribonuclease I were respectively added, and the reaction was stirred for 13 hours, and the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
2)氧化：控制步骤1)所得第一滤液温度至45℃，用20％(w/v)氢氧化钠溶液调整第一滤液pH值至10.0～10.5，加入5μl叔丁基过氧化氢，搅拌反应8小时，得第一纯化液。2) Oxidation: control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 10.0 to 10.5 with 20% (w/v) sodium hydroxide solution, add 5 μl of t-butyl hydroperoxide, and stir. The reaction was carried out for 8 hours to obtain a first purified liquid.
3)超滤除杂：采用截留分子量为4000Da的超滤膜对步骤2)所得第一纯化液进行切向流循环超滤，循环超滤8小时，收集得第一截留液160μl。3) Ultrafiltration removal: The first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cutoff of 4000 Da, and subjected to ultrafiltration for 8 hours, and 160 μl of the first retentate was collected.
4)醇沉除杂：向步骤3)所得的第一截留液中加入130μl甲醇，沉淀16小时，分离得初级沉淀物；再将初级沉淀物加水溶解至160μl，并加入180μl甲醇，沉淀17小时，分离得第一沉淀物，将第一沉淀物真空干燥并称重(36.4mg)。4) Alcohol precipitation: Add 130 μl of methanol to the first retentate obtained in step 3), precipitate for 16 hours, and separate the primary precipitate; then dissolve the primary precipitate into water to 160 μl, add 180 μl of methanol, and precipitate for 17 hours. The first precipitate was isolated and the first precipitate was dried in vacuo and weighed (36.4 mg).
5)降解：向步骤4)所得第一沉淀物中加280mg水溶解，用2mol/L盐酸溶液调节pH值至3.0～3.5，调整温度至25℃，加入0.78mg亚硝酸钠，搅拌反应100分钟，得到第一料液。5) Degradation: add 280 mg of water to the first precipitate obtained in step 4), adjust the pH to 3.0-3.5 with 2 mol/L hydrochloric acid solution, adjust the temperature to 25 ° C, add 0.78 mg of sodium nitrite, and stir the reaction for 100 minutes. , the first liquid is obtained.
6)还原：降解反应完成后，迅速用20％(w/v)的氢氧化钠溶液调节第一料液的pH值至9.5～10.0，加入0.36mg硼氢化钾，搅拌反应12小时，得到第二料液。反应完成后用4mol/L的盐酸溶液调节第二料液的pH值至4.0～5.0，继续搅拌反应20分钟，得到第三料液。反应完成后，再用20％(w/v)的氢氧化钠溶液调节第三料液的pH值至7.5～8.0，并加入1.0％(w/v)第三料液体积的NaCl，搅拌溶解并过滤得到第二滤液，向第二滤液中加入4倍体积的药用乙醇，搅拌后静置13小时，去上清液，得到第二沉淀物。向第二沉淀物中加入水至260μl，搅拌使其溶解，并进行30分钟紫外照射，得到第四料液。6) Reduction: After the degradation reaction is completed, the pH of the first liquid is adjusted to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, 0.36 mg of potassium borohydride is added, and the reaction is stirred for 12 hours to obtain the first Two liquids. After the completion of the reaction, the pH of the second liquid was adjusted to 4.0 to 5.0 with a 4 mol/L hydrochloric acid solution, and the reaction was further stirred for 20 minutes to obtain a third liquid. After the reaction is completed, the pH of the third liquid is adjusted to 7.5-8.0 with 20% (w/v) sodium hydroxide solution, and 1.0% (w/v) of the third liquid volume of NaCl is added and stirred to dissolve. The second filtrate was filtered, and 4 volumes of medicinal ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 13 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 260 μl, stirred to dissolve, and ultraviolet irradiation was carried out for 30 minutes to obtain a fourth liquid.
7)碱氧纯化：用20％(w/v)的氢氧化钠溶液调节第四料液的pH值至9.5～10.0，加入3％第四料液体积的过氧化氢，30℃的温度下搅拌反应8小时，得到第五料液。氧化完毕后，用1mol/L的盐酸溶液调节第五料液pH至7.5～8.0，向其中加入2％(w/v)第五料液体积的NaCl及2.5倍第五料液体积的乙醇，搅拌25分钟以后，静置沉淀9小时，去上清液，得到第三沉淀物，将第三沉淀物用160mg水溶解，得到第二纯化液。7) Alkaline oxygen purification: adjust the pH of the fourth liquid to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, add 3% of the fourth liquid volume of hydrogen peroxide, at a temperature of 30 ° C The reaction was stirred for 8 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth solution is adjusted to 7.5-8.0 with a 1 mol/L hydrochloric acid solution, and 2% (w/v) of the fifth solution volume of NaCl and 2.5 times of the fifth solution volume of ethanol are added thereto. After stirring for 25 minutes, the precipitate was allowed to stand for 9 hours, and the supernatant was removed to obtain a third precipitate, and the third precipitate was dissolved in 160 mg of water to obtain a second purified solution.
8)超滤精制：将步骤7)所得第二纯化液用5000Da的膜循环超滤，收集得到第二截留液：第二截留液经0.25μm的膜超滤，得第三滤液，调节第三滤液pH值至7～7.4。8) Ultrafiltration purification: the second purification liquid obtained in the step 7) is subjected to ultrafiltration through a 5000 Da membrane, and the second retentate is collected: the second retentate is ultrafiltered through a 0.25 μm membrane to obtain a third filtrate, and the third adjustment is performed. The filtrate has a pH of 7 to 7.4.
9)冻干：将调节pH后的第三滤液冻干，得低分子肝素达肝素钠标准品库19.6mg。9) Lyophilization: The third filtrate after pH adjustment was lyophilized to obtain 19.6 mg of a low molecular weight heparin heparin sodium standard library.
实施例6：Example 6
具体步骤：Specific steps:
1)酶解：称取粗品肝素钠40mg，向其中加入460mg水，溶解得原料液；调节原料液温度至36℃，分别加入木瓜蛋白酶0.72mg、核糖核酸酶II 0.32mg及脱氧核糖核酸酶I 0.2mg，搅拌反应12小时，去沉淀，上清液过滤，收集得第一滤液。 1) Enzymatic hydrolysis: 40 mg of crude heparin sodium was weighed, 460 mg of water was added thereto, and the raw material liquid was dissolved; the temperature of the raw material liquid was adjusted to 36 ° C, and 0.53 mg of papain, 0.32 mg of ribonuclease II, and deoxyribonuclease I were respectively added. 0.2 mg, the reaction was stirred for 12 hours, the precipitate was removed, and the supernatant was filtered to collect the first filtrate.
2)氧化：控制步骤1)所得第一滤液温度至45℃，用20％(w/v)氢氧化钠溶液调整第一滤液pH值至9.5～10.0，加入8mg过氧化钠，搅拌反应8小时，得第一纯化液。2) Oxidation: control step 1) to obtain a temperature of the first filtrate to 45 ° C, adjust the pH of the first filtrate to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, add 8 mg of sodium peroxide, and stir the reaction for 8 hours. The first purified liquid was obtained.
3)超滤除杂：采用截留分子量为3000Da的超滤膜对步骤2)所得第一纯化液进行切向流循环超滤，循环超滤10小时，收集得第一截留液180μl。3) Ultrafiltration removal: The first purification liquid obtained in the step 2) was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 3000 Da, and subjected to ultrafiltration for 10 hours, and 180 μl of the first retentate was collected.
4)醇沉除杂：向步骤3)所得的第一截留液中加入140μl甲醇，沉淀15小时，分离得初级沉淀物；再将初级沉淀物加水溶解至180μl，并加入180μl甲醇，沉淀16小时，分离得第一沉淀物，将第一沉淀物真空干燥并称重(35.6mg)。4) Alcohol precipitation: Add 140 μl of methanol to the first retentate obtained in step 3), precipitate for 15 hours, and separate the primary precipitate; then dissolve the primary precipitate into water to 180 μl, add 180 μl of methanol, and precipitate for 16 hours. The first precipitate was isolated, and the first precipitate was vacuum dried and weighed (35.6 mg).
5)降解：向步骤4)所得第一沉淀物中加260mg水溶解，用2mol/L盐酸溶液调节pH值至2.0～2.5，调整温度至20℃，加入0.76mg亚硝酸钠，搅拌反应90分钟，得到第一料液。5) Degradation: 260 mg of water was added to the first precipitate obtained in step 4), the pH was adjusted to 2.0 to 2.5 with 2 mol/L hydrochloric acid solution, the temperature was adjusted to 20 ° C, 0.76 mg of sodium nitrite was added, and the reaction was stirred for 90 minutes. , the first liquid is obtained.
6)还原：降解反应完成后，迅速用20％(w/v)的氢氧化钠溶液调节第一料液的pH值至9.5～10.0，加入0.37mg氢化铝锂，搅拌反应12小时，得到第二料液。反应完成后用4mol/L的盐酸溶液调节第二料液的pH值至4.0～5.0，继续搅拌反应20分钟，得到第三料液。反应完成后，再用20％(w/v)的氢氧化钠溶液调节第三料液的pH值至7.5～8.0，并加入1.0％(w/v)第三料液体积的NaCl，搅拌溶解并过滤得到第二滤液，向第二滤液中加入4倍体积的药用乙醇，搅拌后静置13小时，去上清液，得到第二沉淀物。向第二沉淀物中加入水至260μl，搅拌使其溶解，并进行30分钟紫外照射，得到第四料液。6) Reduction: After the degradation reaction is completed, the pH of the first liquid is adjusted to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, 0.37 mg of lithium aluminum hydride is added, and the reaction is stirred for 12 hours to obtain the first Two liquids. After the completion of the reaction, the pH of the second liquid was adjusted to 4.0 to 5.0 with a 4 mol/L hydrochloric acid solution, and the reaction was further stirred for 20 minutes to obtain a third liquid. After the reaction is completed, the pH of the third liquid is adjusted to 7.5-8.0 with 20% (w/v) sodium hydroxide solution, and 1.0% (w/v) of the third liquid volume of NaCl is added and stirred to dissolve. The second filtrate was filtered, and 4 volumes of medicinal ethanol was added to the second filtrate. After stirring, the mixture was allowed to stand for 13 hours, and the supernatant was removed to obtain a second precipitate. Water was added to the second precipitate to 260 μl, stirred to dissolve, and ultraviolet irradiation was carried out for 30 minutes to obtain a fourth liquid.
7)碱氧纯化：用20％(w/v)的氢氧化钠溶液调节第四料液的pH值至9.5～10.0，加入3％第四料液体积的叔丁基过氧化氢，30℃的温度下搅拌反应8小时，得到第五料液。氧化完毕后，用1mol/L的盐酸溶液调节第五料液pH至7.5～8.0，向其中加入3％(w/v)第五料液体积的NaCl及3倍第五料液体积的乙醇，搅拌25分钟以后，静置沉淀10小时，去上清液，得到第三沉淀物，将第三沉淀物用200mg水溶解，得到第二纯化液。7) Alkaline oxygen purification: adjust the pH of the fourth liquid to 9.5 to 10.0 with 20% (w/v) sodium hydroxide solution, add 3% of the fourth liquid volume of t-butyl hydroperoxide, 30 ° C The reaction was stirred at a temperature of 8 hours to obtain a fifth liquid. After the oxidation is completed, the pH of the fifth liquid solution is adjusted to 7.5-8.0 with a 1 mol/L hydrochloric acid solution, and 3% (w/v) of the fifth liquid volume of NaCl and 3 times of the fifth liquid volume of ethanol are added thereto. After stirring for 25 minutes, the precipitate was allowed to stand for 10 hours, and the supernatant was removed to obtain a third precipitate, and the third precipitate was dissolved in 200 mg of water to obtain a second purified solution.
8)超滤精制：将步骤7)所得第二纯化液用4000Da的膜循环超滤，收集得到第二截留液：第二截留液经0.2μm的膜超滤，得第三滤液，调节第三滤液pH值至6.5～7.0。8) Ultrafiltration purification: the second purified liquid obtained in the step 7) is subjected to ultrafiltration through a 4000 Da membrane, and the second retentate is collected to obtain a second retentate. The second retentate is ultrafiltered through a membrane of 0.2 μm to obtain a third filtrate, and the third filtrate is adjusted. The filtrate pH was between 6.5 and 7.0.
9)冻干：将调节pH后的第三滤液冻干，得低分子肝素达肝素钠标准品库16.8mg。9) Lyophilization: The third filtrate after pH adjustment was lyophilized to obtain 16.8 mg of a low molecular weight heparin sodium heparin standard library.
试验例：参照美国药典方法对样品进行活性检测Test example: Activity detection of samples by reference to the United States Pharmacopoeia method
以制备实施例1～6所得低分子肝素达肝素钠标准品库为样品进行试验，试验结果见表一。The low molecular weight heparin sodium heparin standard library obtained in Preparation Examples 1 to 6 was used as a sample, and the test results are shown in Table 1.
表一：Table I:
Figure PCTCN2017090902-appb-000001
Figure PCTCN2017090902-appb-000001
Figure PCTCN2017090902-appb-000002
Figure PCTCN2017090902-appb-000002
Figure PCTCN2017090902-appb-000003
Figure PCTCN2017090902-appb-000003
由上述检测结果可知，本方法生产的低分子肝素达肝素钠标准品库符合欧洲药典标准，其平均分子量更接近于6000Da的特征值，且抗血栓活性(抗FXa)优于市场上销售的同类产品。It can be seen from the above test results that the low molecular weight heparin sodium heparin standard library produced by the method conforms to the European Pharmacopoeia standard, the average molecular weight is closer to the characteristic value of 6000 Da, and the antithrombotic activity (anti-FXa) is superior to the similar products on the market. product.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, and are not intended to be limiting; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art The technical solutions described in the foregoing embodiments may be modified, or some or all of the technical features may be equivalently replaced; and the modifications or substitutions do not deviate from the technical solutions of the embodiments of the present invention. The scope.
本发明的低分子肝素达肝素钠标准品库的制备方法具有以下突出的有益效果：The preparation method of the low molecular weight heparin sodium heparin standard library of the invention has the following outstanding beneficial effects:
(一)以复合酶解和改良的亚硝酸盐降解法为基础，以粗品肝素钠为原料，选用木瓜蛋白酶、核糖核酸酶II等蛋白和核酸酶优化组合，对粗品肝素中蛋白和核酸进行高效降解，并结合膜技术一次性去除蛋白和核酸杂质，避免了传统工艺中从粗品到精品制备反复除杂的繁琐过程；(1) Based on the composite enzymatic hydrolysis and modified nitrite degradation method, using crude heparin sodium as raw material, using papain, ribonuclease II and other proteins and nucleases to optimize the combination of proteins and nucleic acids in crude heparin Degradation, combined with membrane technology to remove protein and nucleic acid impurities at one time, avoiding the cumbersome process of repeated decontamination from crude to fine preparation in traditional processes;
(二)未分级肝素在特定的温度和pH条件下，与亚硝酸盐作用，使未分级肝素中的N-硫酸葡萄糖胺反应脱去HSO4-形成-NH2，-NH2与亚硝酸盐发生重氮化反应，在放出N2的同时糖苷键断裂，电子转移，缩环生成2,5-脱氢甘露醛，后者经还原糖或脱氢形成甘露糖醇，后通过低分子膜分离技术获得具有特定平均分子量(5800-6200)的低分子肝素达肝素钠标准品库；(2) Unfractionated heparin reacts with nitrite at a specific temperature and pH to desorb HSO 4 in the unfractionated heparin to form HSO 4 - form -NH 2 , -NH 2 and nitrite The diazotization reaction occurs, the glycosidic bond is broken while the N 2 is released, and the electron transfer and condensed ring form 2,5-dehydromannuraldehyde, which is formed by reducing sugar or dehydrogenation to form mannitol, and then separated by a low molecular membrane. The technology obtains a library of low molecular weight heparin sodium heparin standards with a specific average molecular weight (5800-6200);
(三)通过本发明方法制备得到的低分子肝素达肝素钠标准品库平均分子量为5800～6200，分子量分布：＜1600的比例≤40.0％；＞4500的比例≤11.0％；抗FXa/FIIa＞30，抗FXa效价：195～210IU/mg，抗FIIa效价：＜5.0IU/mg，与其他低分子肝素钠相比，有着非常好的稳定性和抗血栓活性，产品质量超越欧洲药典标准，有望成为新生代的肝素类抗血栓药物。(3) The average molecular weight of the low molecular weight heparin sodium heparin standard library prepared by the method of the invention is 5800-6200, the molecular weight distribution: <1600 ratio ≤ 40.0%; > 4500 ratio ≤ 11.0%; anti-FXa/FIIa> 30, anti-FXa titer: 195 ~ 210IU / mg, anti-FIIa titer: <5.0IU / mg, compared with other low molecular weight heparin sodium, has very good stability and antithrombotic activity, product quality beyond the European Pharmacopoeia standards It is expected to become a new generation of heparin antithrombotic drugs.
(四)本发明所提供的低分子肝素达肝素钠标准品库，其分子量为5800Da～6200Da，分子量分布：＜1600的比例≤40.0％；＞4500的比例≤11.0％。具有纯度高、分子量分布集中等优点，达到科研活动中的对于标准品的严苛要求，非常时候作为科研活动中的标准品使用。 (4) The low molecular weight heparin sodium heparin standard library provided by the invention has a molecular weight of 5800 Da to 6200 Da, a molecular weight distribution: <1600 ratio ≤ 40.0%; > 4500 ratio ≤ 11.0%. It has the advantages of high purity, concentrated molecular weight distribution, and meets the stringent requirements for standard products in scientific research activities. It is often used as a standard in scientific research activities.

Claims (18)

  1. 一种低分子肝素达肝素钠标准品库的制备方法，其特征在于，包括以下步骤：A preparation method of a low molecular weight heparin sodium heparin standard library, characterized in that the method comprises the following steps:
    1)酶解：向粗品肝素钠中加入10～15倍质量的水，溶解得原料液，调节所述原料液温度至35℃～39℃，加入复合酶，搅拌反应10～15小时，去沉淀，上清液过滤，收集得到第一滤液；所述复合酶包括木瓜蛋白酶、核糖核酸酶II及脱氧核糖核酸酶I，其中，所述木瓜蛋白酶的用量为所述粗品肝素钠重量的1％～2％，所述核糖核酸酶II的用量为所述粗品肝素钠重量的0.5％～1％，所述脱氧核糖核酸酶I的用量为所述粗品肝素钠重量的0.5％～1％；1) Enzymatic hydrolysis: 10 to 15 times the mass of water is added to the crude heparin sodium, the raw material liquid is dissolved, the temperature of the raw material liquid is adjusted to 35 ° C to 39 ° C, the complex enzyme is added, and the reaction is stirred for 10 to 15 hours to precipitate. The supernatant is filtered, and the first filtrate is collected; the complex enzyme includes papain, ribonuclease II and deoxyribonuclease I, wherein the papain is used in an amount of 1% by weight of the crude heparin sodium. 2%, the ribonuclease II is used in an amount of 0.5% to 1% by weight of the crude heparin sodium, and the amount of the DNase I is 0.5% to 1% by weight of the crude heparin sodium;
    2)氧化：控制所述第一滤液温度至40℃～50℃，并调整所述第一滤液pH值至9～11，加入0.1％～0.5％第一滤液体积的过氧化氢，搅拌反应6～8小时，得第一纯化液；2) oxidation: controlling the temperature of the first filtrate to 40 ° C ~ 50 ° C, and adjusting the pH of the first filtrate to 9 ~ 11, adding 0.1% ~ 0.5% of the first filtrate volume of hydrogen peroxide, stirring reaction 6 ~ 8 hours, the first purified liquid;
    3)超滤除杂：采用截留分子量为3000Da～5000Da的超滤膜对所述第一纯化液进行切向流循环超滤，循环超滤5～10小时，收集得到第一截留液；3) ultrafiltration removal: the first purification liquid is subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 3000 Da to 5000 Da, and circulating ultrafiltration for 5 to 10 hours to collect the first retentate;
    4)醇沉除杂：所述第一截留液醇沉除杂，得到第一沉淀物，对所述第一沉淀物干燥称重；4) alcohol precipitation: the first retentate alcohol is removed to obtain impurities, a first precipitate is obtained, and the first precipitate is dried and weighed;
    5)降解：向所得第一沉淀物中加7～8倍质量的水溶解，用盐酸溶液调节pH值至2.0～4.0，调整温度至15～30℃，加入适量亚硝酸钠，搅拌反应90～120分钟，得到第一料液；所述亚硝酸钠的加入量为所述第一沉淀物质量的2％～3％；5) Degradation: Add 7-8 times of mass of water to the obtained first precipitate to dissolve, adjust the pH value to 2.0-4.0 with hydrochloric acid solution, adjust the temperature to 15-30 °C, add appropriate amount of sodium nitrite, and stir the reaction 90~ 120 minutes, the first liquid solution is obtained; the sodium nitrite is added in an amount of 2% to 3% of the mass of the first precipitate;
    6)还原：降解反应完成后，迅速用氢氧化钠溶液调节所述第一料液的pH值至9.0～10.0，加入适量硼氢化钠，搅拌反应10～15小时，得到第二料液；反应完成后用盐酸溶液调节所述第二料液的pH值至2.0～5.0，继续搅拌反应15～30分钟，得到第三料液；反应完成后，用氢氧化钠溶液调节所述第三料液的pH值至6～8，并加入适量的氯化钠，搅拌溶解并过滤，得到第二滤液，向所述第二滤液中加入3～5倍体积的醇液，搅拌后静置12～15小时；去上清液，得到第二沉淀物，向所述第二沉淀物中加入水，搅拌使其溶解，并进行20～30分钟紫外照射，得到第四料液；所述硼氢化钠的加入量为所述第一沉淀物质量的0.9％～1.1％；氯化钠与所述第三料液的质量体积比为0.9％～1.1％；6) reduction: after the completion of the degradation reaction, rapidly adjust the pH of the first liquid to 9.0 to 10.0 with sodium hydroxide solution, add an appropriate amount of sodium borohydride, and stir the reaction for 10 to 15 hours to obtain a second liquid; After completion, the pH of the second liquid is adjusted to 2.0 to 5.0 with a hydrochloric acid solution, and the reaction is further stirred for 15 to 30 minutes to obtain a third liquid; after the reaction is completed, the third liquid is adjusted with a sodium hydroxide solution. The pH value is 6-8, and an appropriate amount of sodium chloride is added, stirred and dissolved, and filtered to obtain a second filtrate, and 3 to 5 volumes of the alcohol solution is added to the second filtrate, and the mixture is allowed to stand for 12 to 15 after stirring. Hour; remove the supernatant to obtain a second precipitate, add water to the second precipitate, stir to dissolve, and perform ultraviolet irradiation for 20 to 30 minutes to obtain a fourth liquid; the sodium borohydride The amount of addition is 0.9% to 1.1% of the mass of the first precipitate; the mass to volume ratio of sodium chloride to the third liquid is 0.9% to 1.1%;
    7)碱氧纯化：用氢氧化钠溶液调节所述第四料液pH值至9～10，加入1～3％所述第四料液体积的过氧化氢，20～30℃温度下搅拌反应6～8小时，得到所述第五料液；对所述第五料液醇沉，得到第三沉淀物；并将所述第三沉淀物用适量水溶解得到第二纯化液，所用水的量为所述第三沉淀物质量的3～4倍；7) Purification of alkali oxygen: adjust the pH of the fourth liquid to 9-10 with sodium hydroxide solution, add 1 to 3% of the fourth liquid volume of hydrogen peroxide, and stir the reaction at 20-30 °C 6 to 8 hours, the fifth liquid solution is obtained; the fifth liquid solution is alcohol-elected to obtain a third precipitate; and the third precipitate is dissolved with an appropriate amount of water to obtain a second purified liquid, which is used for water. The amount is 3 to 4 times the mass of the third precipitate;
    8)超滤精制：将步骤所述第二纯化液用5000Da～6000Da的膜循环超滤，收集得到第二截留液；所述第二截留液经0.22μm的膜超滤，得第三滤液，调节所述第三滤液 pH值至6.4～7.4；8) ultrafiltration purification: the second purification liquid in the step is ultrafiltered with a membrane of 5000 Da to 6000 Da, and the second retentate is collected; the second retentate is ultrafiltered through a membrane of 0.22 μm to obtain a third filtrate. Adjusting the third filtrate pH value to 6.4 to 7.4;
    9)冻干：将调节pH值后的所述第三滤液冻干，得低分子肝素达肝素钠标准品库。9) Lyophilization: The third filtrate after adjusting the pH value is lyophilized to obtain a low molecular weight heparin heparin sodium standard library.
  2. 根据权利要求1所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，步骤4)醇沉除杂时，向所述第一截留液中加入0.6～0.8倍体积的甲醇，沉淀14～18小时，分离得初级沉淀物；再将所述初级沉淀物加水溶解得到初级沉淀物溶液，向所述初级沉淀物溶液中加入1～1.2倍体积的甲醇，沉淀14～18小时，分离得所述第一沉淀物，将所述第一沉淀物真空干燥并称重。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 1, wherein in step 4), when the alcohol is removed, 0.6 to 0.8 times of methanol is added to the first retentate. Precipitating for 14 to 18 hours, separating the primary precipitate; then adding the primary precipitate to water to obtain a primary precipitate solution, adding 1 to 1.2 volumes of methanol to the primary precipitate solution, and precipitating for 14 to 18 hours. The first precipitate was separated, and the first precipitate was vacuum dried and weighed.
  3. 根据权利要求1所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，步骤6)中，所述醇液为甲醇溶液或乙醇溶液。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 1, wherein in the step 6), the alcohol liquid is a methanol solution or an ethanol solution.
  4. 根据权利要求1所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，步骤7)醇沉的具体过程为：用盐酸溶液调节所述第五料液的pH值至6～8，加入适量氯化钠及2～3倍体积的乙醇，搅拌20～30分钟以后，静置沉淀8～10小时，去上清液；氯化钠与所述第五料液的质量体积比为1％～3％。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 1, wherein the step 7) the alcohol precipitation is carried out by adjusting the pH of the fifth liquid to 6 to 6 with a hydrochloric acid solution. 8, adding appropriate amount of sodium chloride and 2 to 3 volumes of ethanol, stirring for 20 to 30 minutes, standing precipitation for 8 to 10 hours, removing the supernatant; mass to volume ratio of sodium chloride to the fifth liquid It is 1% to 3%.
  5. 一种低分子肝素达肝素钠标准品库的制备方法，其特征在于，包括以下步骤：A preparation method of a low molecular weight heparin sodium heparin standard library, characterized in that the method comprises the following steps:
    将粗品肝素钠的溶液与复合酶混合反应，过滤后得到第一滤液；Mixing a solution of crude heparin sodium with a complex enzyme, and filtering to obtain a first filtrate;
    将所述第一滤液调整为碱性，与氧化剂混合发生氧化反应，醇沉后得到第一沉淀物；Adjusting the first filtrate to be alkaline, mixing with an oxidant to generate an oxidation reaction, and obtaining a first precipitate after alcohol precipitation;
    将所述第一沉淀物溶解，并调整第一沉淀物的溶液为酸性，与亚硝酸盐混合降解，得到降解液；Dissolving the first precipitate, adjusting the solution of the first precipitate to be acidic, and degrading with nitrite to obtain a degradation liquid;
    所述降解液与还原剂混合发生还原反应。The degradation solution is mixed with a reducing agent to cause a reduction reaction.
  6. 根据权利要求5所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，所述复合酶包括木瓜蛋白酶、核糖核酸酶II和脱氧核糖核酸酶I；其中，所述木瓜蛋白酶的用量为所述粗品肝素钠重量的1％～2％，优选为1.2％～1.8％，所述核糖核酸酶II的用量为所述粗品肝素钠重量的0.5％～1％，优选为0.5％～0.8％，所述脱氧核糖核酸酶I的用量为所述粗品肝素钠重量的0.5％～1％，优选为0.5％～0.8％。The method for preparing a low molecular weight heparin heparin sodium standard library according to claim 5, wherein the complex enzyme comprises papain, ribonuclease II and deoxyribonuclease I; wherein the papain The amount is 1% to 2%, preferably 1.2% to 1.8%, based on the weight of the crude heparin sodium, and the ribonuclease II is used in an amount of 0.5% to 1%, preferably 0.5% by weight of the crude heparin sodium. 0.8%, the amount of the DNase I is from 0.5% to 1%, preferably from 0.5% to 0.8%, based on the weight of the crude heparin sodium.
  7. 根据权利要求5或6所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，所述粗品肝素钠的溶液与所述复合酶混合反应的温度为35℃～39℃，反应时间为10～15小时。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 5 or 6, wherein the temperature of the mixed solution of the crude heparin sodium and the complex enzyme is 35 ° C to 39 ° C, and the reaction The time is 10 to 15 hours.
  8. 根据权利要求5所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，在所述第一滤液在与所述氧化剂反应之前，将所述第一滤液的pH值调整至9～11，温度调整至40℃～50℃。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 5, wherein the pH of the first filtrate is adjusted to 9 before the first filtrate reacts with the oxidizing agent. ~11, the temperature is adjusted to 40 ° C ~ 50 ° C.
  9. 根据权利要求5或8所述的低分子肝素达肝素钠标准品库的制备方法，其特征 在于，所述氧化剂选自由过氧化氢、叔丁基过氧化氢、过氧化钠和过氧化钾组成的组中的至少一种，优选地，所述氧化剂选自由过氧化氢、叔丁基过氧化氢组成的组中的至少一种，更优选地，所述氧化剂为过氧化氢，所述氧化剂的体积为所述第一滤液的0.1％～0.5％，优选为0.2％～0.3％。A method for preparing a low molecular weight heparin heparin sodium standard library according to claim 5 or 8, characterized in that The oxidizing agent is at least one selected from the group consisting of hydrogen peroxide, t-butyl hydroperoxide, sodium peroxide, and potassium peroxide. Preferably, the oxidizing agent is selected from the group consisting of hydrogen peroxide and t-butyl. At least one of the group consisting of hydrogen peroxide, more preferably, the oxidizing agent is hydrogen peroxide, and the volume of the oxidizing agent is 0.1% to 0.5%, preferably 0.2% to 0.3%, of the first filtrate.
  10. 根据权利要求5～9任一项所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，所述第一滤液在与所述氧化剂混合发生氧化反应之后，醇沉之前还包括第一次除杂步骤：将所述第一滤液与所述氧化剂混合反应之后得到的纯化液，采用截留分子量为3000Da～5000Da的超滤膜进行切向流循环超滤，超滤时间为5～10小时，得到第一截留液。The method for preparing a low molecular weight heparin sodium heparin standard library according to any one of claims 5 to 9, wherein the first filtrate is mixed with the oxidizing agent to form an oxidation reaction, and before the alcohol precipitation The first impurity removing step: the purified liquid obtained by mixing the first filtrate and the oxidizing agent is subjected to tangential flow circulating ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 3000 Da to 5000 Da, and the ultrafiltration time is 5 ～. After 10 hours, the first retentate was obtained.
  11. 根据权利要求10所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，将所述第一截留液与醇溶剂混合，沉淀14～18小时，分离得到所述第一沉淀物；所述第一截留液与醇溶剂的体积比为1：0.6～0.8。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 10, wherein the first retentate is mixed with an alcohol solvent, and precipitated for 14 to 18 hours to separate the first precipitate. The volume ratio of the first retentate to the alcohol solvent is 1:0.6-0.8.
  12. 根据权利要求5所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，在将所述第一沉淀物的溶液与所述亚硝酸盐混合降解之前，将所述第一沉淀物的溶液的pH值调整为2.0～4.0，温度调整为15℃～30℃；所述第一沉淀物的溶液与所述亚硝酸盐混合降解的反应时间为90～120分钟，所述亚硝酸盐的质量为所述第一沉淀物的2％～3％。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 5, wherein the first precipitate is formed before the solution of the first precipitate is mixed with the nitrite to be degraded The pH of the solution of the solution is adjusted to 2.0 to 4.0, and the temperature is adjusted to 15 ° C to 30 ° C; the reaction time of the solution of the first precipitate mixed with the nitrite is 90 to 120 minutes, and the nitrous acid The mass of the salt is from 2% to 3% of the first precipitate.
  13. 根据权利要求12所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，所述还原剂选自由硼氢化钠、硼氢化钾和氢化铝锂组成的组中的至少一种，优选为硼氢化钠或硼氢化钾，更优选为硼氢化钠。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 12, wherein the reducing agent is at least one selected from the group consisting of sodium borohydride, potassium borohydride and lithium aluminum hydride. It is preferably sodium borohydride or potassium borohydride, more preferably sodium borohydride.
  14. 根据权利要求12或13所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，所述降解液与所述还原剂反应之前，先将所述降解液的pH值调整至9.0～10.0；所述还原剂的质量为所述第一沉淀物的0.9％～1.1％；所述降解液与所述还原剂反应的时间为10～15小时。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 12 or 13, wherein the pH of the degradation liquid is adjusted to 9.0 before the degradation liquid reacts with the reducing agent. ～10.0; the mass of the reducing agent is 0.9% to 1.1% of the first precipitate; and the time for the degradation liquid to react with the reducing agent is 10 to 15 hours.
  15. 根据权利要求5～14任一项所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，还包括所述降解液与所述还原剂混合发生还原反应之后的后处理步骤：将所述降解液和所述还原剂反应之后得到的反应液的pH值调整至2.0～5.0，反应15～30分钟，再调整pH至6～8，过滤得到第二滤液；将所述第二滤液与3～5倍体积的醇溶剂混合，静置12～15小时，过滤得到第二沉淀物；将所述第二沉淀物溶解，并将所述第二沉淀物的溶液进行20～30分钟的紫外照射。The method for preparing a low molecular weight heparin heparin sodium standard library according to any one of claims 5 to 14, further comprising a post-processing step after the degradation solution is mixed with the reducing agent to cause a reduction reaction: Adjusting the pH of the reaction liquid obtained after the reaction of the degradation liquid and the reducing agent to 2.0 to 5.0, reacting for 15 to 30 minutes, adjusting the pH to 6 to 8, and filtering to obtain a second filtrate; The filtrate is mixed with 3 to 5 volumes of the alcohol solvent, allowed to stand for 12 to 15 hours, and filtered to obtain a second precipitate; the second precipitate is dissolved, and the solution of the second precipitate is allowed to be carried out for 20 to 30 minutes. UV irradiation.
  16. 根据权利要求15所述的低分子肝素达肝素钠标准品库的制备方法，其特征在于，还包括后处理步骤之后的第二次除杂步骤：将紫外照射后的所述第二沉淀物的溶 液进行氧化、醇沉，并采用5000Da～6000Da的膜循环超滤，得到第二截留液，将所述第二截留液经0.2～0.25μm的膜超滤得到第三滤液，将所述第三滤液的pH调整至6.4～7.4，并进行冻干处理。The method for preparing a low molecular weight heparin sodium heparin standard library according to claim 15, further comprising a second impurity removing step after the post-treatment step: the second precipitate after ultraviolet irradiation Dissolve The liquid is subjected to oxidation, alcohol precipitation, and ultrafiltration through a membrane of 5000 Da to 6000 Da to obtain a second retentate, and the second retentate is ultrafiltered through a membrane of 0.2 to 0.25 μm to obtain a third filtrate, and the third The pH of the filtrate was adjusted to 6.4 to 7.4 and lyophilized.
  17. 一种低分子肝素达肝素钠标准品库，其特征在于，由如权利要求1～16任一项所述的低分子肝素达肝素钠标准品库的制备方法制备得到，所述低分子肝素达肝素钠标准品库的分子量为5800Da～6200Da，分子量分布：＜1600的比例≤40.0％；＞4500的比例≤11.0％。A low molecular weight heparin sodium heparin standard library, which is prepared by the preparation method of the low molecular weight heparin heparin sodium standard library according to any one of claims 1 to 16, wherein the low molecular weight heparin The molecular weight of the heparin sodium standard library is 5800 Da to 6200 Da, and the molecular weight distribution: <1600 ratio ≤ 40.0%; > 4500 ratio ≤ 11.0%.
  18. 根据权利要求17所述的低分子肝素达肝素钠标准品库，其特征在于，所述低分子肝素达肝素钠标准品库的抗FXa/FIIa＞30，抗FXa效价：195～210IU/mg，抗FIIa效价：＜5.0IU/mg。 The low molecular weight heparin sodium heparin standard library according to claim 17, wherein the low molecular weight heparin sodium heparin standard library has anti-FXa/FIIa>30, anti-FXa titer: 195-210 IU/mg , anti-FIIa titer: <5.0IU/mg.
PCT/CN2017/090902 2017-06-29 2017-06-29 Standard library of low-molecular-weight heparin, dalteparin sodium, and preparation method thereof WO2019000335A1 (en)

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