WO2019000336A1 - 低分子肝素那曲肝素钙标准品库及其制备方法 - Google Patents

低分子肝素那曲肝素钙标准品库及其制备方法 Download PDF

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WO2019000336A1
WO2019000336A1 PCT/CN2017/090904 CN2017090904W WO2019000336A1 WO 2019000336 A1 WO2019000336 A1 WO 2019000336A1 CN 2017090904 W CN2017090904 W CN 2017090904W WO 2019000336 A1 WO2019000336 A1 WO 2019000336A1
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precipitate
molecular weight
liquid
calcium
filtrate
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PCT/CN2017/090904
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English (en)
French (fr)
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周霞
雷晓刚
乔德强
郭维
林勇
郭恩中
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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/CN2017/090904 priority Critical patent/WO2019000336A1/zh
Publication of WO2019000336A1 publication Critical patent/WO2019000336A1/zh

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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

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  • the invention relates to the field of biomedicine, in particular to a low molecular weight heparin nadroparin standard library (Nadroparin) 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 low molecular weight heparin nadropar calcium standard library, not only has antithrombotic effect superior to unfractionated heparin, but also has bioavailability. High, long half-life in the body, and low bleeding tendency.
  • anticoagulant activity of heparin is divided into two major categories: antithrombotic activity (FXa) and anticoagulant (FIIa) activity.
  • FXa antithrombotic activity
  • FIIa anticoagulant 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 weight heparin Natraheparin calcium is an anticoagulant drug that is depolymerized by acidic calcium nitrite, reduced by sodium borohydride and UV modified, and replaced by oxidation and calcium. It is a kind of low molecular weight heparin.
  • the preparation methods in the international and domestic are relatively simple, all of which are nitrite degradation, but on this condition, the degradation of the product is unstable, the internal structure is damaged, and the structural confirmation rate is low.
  • the usual preparation method is: dissolving the purchased crude heparin sodium raw material in purified water, directly adding a certain amount of sodium nitrite, controlling the pH value in the range of 2.7 to 2.9, and stirring the reaction at a certain temperature for 25 to 35 minutes.
  • the finished product is reduced, calcium, filtered, concentrated, etc.
  • the traditional method is to purchase the fine heparin as raw material, it is difficult to control the quality of the final product from the source of the raw material.
  • degrading only heparin can be degraded by nitrite to produce low molecular weight, and the degradation process is difficult to control the dramatic change of pH. Uncontrollable molecular weight and structure of the product, and poor product stability.
  • a first object of the present invention is to provide a method for preparing a low molecular weight heparin nadroparin calcium standard library in view of the above-mentioned deficiencies of the prior art.
  • the method overcomes the uncontrollability of the raw materials in the traditional process and the product yield is low, and the product is stable. Defects in poor sex.
  • the quality of the low molecular weight heparin natriuretic calcium standard library produced by this method is higher than the national standard and the European quality standard, and the stability is good, and the product yield is high.
  • a second object of the present invention is to provide a low molecular weight heparin natriuretic calcium standard library prepared by the preparation method of the above low molecular weight heparin nadropar calcium standard library, which has good stability, high bioavailability and half life in vivo. Long, bleeding tendency is small.
  • the method uses crude heparin sodium as a raw material, and uses an optimized combination of protein and nuclease such as papain and ribonuclease II to efficiently degrade protein and nucleic acid in crude heparin sodium, and Combine membrane technology to remove protein and nucleic acid impurities in one go.
  • the treated sodium heparin is completely dissolved in the buffered glacial acetic acid, and reacts with a nitrite such as calcium nitrite under specific temperature and acidic conditions to cause deamination on the N-sulfate glucosamine residue.
  • the rearrangement reaction causes a cleavage of the glycosidic bond between the glucosamine and the uronic acid to form a 2,5-anhydromannose terminal residue, and then reduction treatment to obtain a stable 2,5-anhydromannitol, after ultraviolet irradiation.
  • the nano-molecular weight of the low-molecular-weight heparin natriuretic calcium standard library with a molecular weight of 3800-5000 Daltons is obtained by nanofiltration technology and low molecular membrane separation technology.
  • the crude product of the heparin calcium standard library is decolorized by oxidation by oxidation, ethanol precipitation, and vacuum freeze-drying to obtain anti-FXa factor activity of 100 IU/mg to 130 IU/mg, pH value of 6.5 to 7.2, and anti-FXa/FIIa ratio of 2.7 to 4.0.
  • the finished product of the low molecular weight heparin natrasperm calcium standard product with an average molecular weight of 3800-5000D.
  • an embodiment of the present application provides a method for preparing a low molecular weight heparin natriuretic calcium standard library, comprising the following steps:
  • enzymatic hydrolysis adding 10 to 12 times the mass of water to the crude heparin sodium to dissolve, dissolve the first liquid solution, adjust the pH of the first liquid to 8.0 to 8.5, the temperature to 35 to 40 ° C, and add papain And ribonuclease II, enzymatic hydrolysis reaction for 8 to 10 hours, centrifugation to precipitate, the supernatant is filtered, and the first filtrate is collected;
  • Oxidative decolorization control the temperature of the first filtrate to 30-40 ° C, adjust the pH of the first filtrate to 9-11, add 0.3% to 0.5% of the first filtrate volume of hydrogen peroxide, and stir the reaction for 6-8 hours.
  • ultraviolet irradiation adding 8 to 10 times the mass of water to the second precipitate, stirring, dissolving, and irradiating with ultraviolet rays of a wavelength of 254 nm for 20 to 30 minutes at room temperature to obtain a fifth liquid;
  • oxidation adjusting the pH of the fifth liquid to 9 to 10, adding 0.8% to 1.0% of the volume of hydrogen peroxide, adjusting the temperature of the fifth liquid to 20 to 30 ° C, stirring the reaction for 6 to 8 hours; After oxidation, alcohol precipitation, static precipitation for 8-10 hours, the supernatant is removed to obtain a third precipitate, and 9-10 times of mass of water is added to the third precipitate to dissolve;
  • Nanofiltration After the reaction is completed, the replacement liquid is filtered and added to the nanofiltration machine to start circulating nanofiltration. When the volume concentration of the nanofiltration retentate reaches 20% to 25%, the second retentate is released, and the second retentate volume is added. ⁇ 3 times the amount of 95% ethanol, static precipitation for 12 to 15 hours to obtain a calcium salt precipitate; in the circulating nanofiltration process can add an appropriate amount of purified water;
  • the amount of papain in step a is 0.5% to 0.7% by weight of crude heparin sodium, and the amount of ribonuclease II is 0.3% to 0.5% by weight of crude heparin sodium. %.
  • step d and step h the alcohol precipitation operation can be completed by the alcohol precipitation method well known in the prior art.
  • the following specific methods are preferably implemented:
  • step d when alcohol is removed in step d, sodium chloride is added to the first retentate, and after stirring, 0.8 to 1.0 volume of 95% ethanol is added. Precipitating for 10-12 hours, separating the intermediate precipitate; then dissolving the intermediate precipitate with water, adding 1-1.2 times volume of 95% ethanol to the solution, and precipitating for 6-8 hours, separating the first precipitate, the first precipitate
  • the material is vacuum dried and weighed; the mass to volume ratio of sodium chloride to the first retentate is from 1.0% to 1.2%.
  • the specific process of alcohol precipitation in step h is: adjusting the pH of the replacement liquid to 6-8, adding sodium chloride to the replacement liquid and 2 to 3 times the volume. 95% ethanol; the mass to volume ratio of sodium chloride to the replacement liquid is 0.8% to 1.0%.
  • the nitrite in step e is calcium nitrite.
  • an embodiment of the present application provides a method for preparing a low molecular weight heparin nadropar calcium standard library, which comprises the following steps:
  • the concentration of glacial acetic acid is 1.8 to 2.0%; the amount of glacial acetic acid added is 7 to 8 times the mass of the first precipitate; and the amount of nitrite is the first 1.5% to 2.1% by weight of a precipitate.
  • the method before the first filtrate is subjected to multiple times of decontamination, the method further comprises: performing oxidative decolorization on the first filtrate: controlling the temperature of the first filtrate to 30-40 ° C, adjusting the first The pH of the filtrate is adjusted to 9 to 11, and 0.3% to 0.5% of the first filtrate volume of hydrogen peroxide is added, and the reaction is stirred for 6 to 8 hours to obtain a purified liquid.
  • the plurality of first filtrates are removed: the pH of the purification solution is adjusted to 6-8, and the purification liquid is subjected to an ultrafiltration membrane having a molecular weight cut off of 3,000 to 8,000 Da.
  • the step of reducing the degradation liquid is further included: adjusting the pH of the degradation solution to 9.0 to 10.0, adding sodium borohydride, and stirring the reaction for 10 to 16 hours. Adjust the pH to 6-8, then add sodium chloride, stir to dissolve, add ethanol to the filtrate after filtration, stir for 20 to 30 minutes, then let stand for 8 to 12 hours; remove the supernatant to obtain a second precipitate.
  • the method further comprises: after the second precipitate is subjected to ultraviolet irradiation, oxidation, and calcium replacement, the replacement liquid is obtained, and the replacement liquid is filtered.
  • the second precipitate is subjected to ultraviolet irradiation, oxidation, and calcium replacement, the replacement liquid is obtained, and the replacement liquid is filtered.
  • the method further comprises: adding 10 to 12 times of mass of water to the calcium salt precipitate for dissolution, and completely dissolving 0.2-
  • the membrane was filtered by 0.3 um, then the molecular weight was controlled by ultrafiltration of 1000-3000 Da membrane, and the third retentate was collected; the third retentate was filtered through a 0.1-0.2 ⁇ m decontamination membrane to collect the third filtrate, and the third was adjusted.
  • the pH of the filtrate was adjusted to 6.5 to 7.2; the third filtrate was subjected to lyophilization.
  • the embodiment of the present application further provides a low molecular weight heparin nadropar calcium standard library, which is prepared by the preparation method of the low molecular weight heparin nadropar calcium standard library.
  • the low molecular weight heparin natriuretic calcium standard library has an average molecular weight of from 3,800 to 5,000 Daltons.
  • the molecular weight distribution of the low molecular weight heparin nadropar calcium standard library is: a ratio of molecular weight less than 2000 is ⁇ 15.0%; a ratio of molecular weight of 2000 to 8000 is 75.0% ⁇ 95.0%; the ratio of the molecular weight in the range of 2000 to 4000 is 35.0% to 55.0%.
  • the low molecular weight heparin nadropar calcium standard library has an anti-FXa factor activity of 100 IU/mg to 130 IU/mg, a pH of 6.5 to 7.2, and an anti-FXa/FIIa ratio. It is 2.7 to 4.0.
  • the low molecular weight heparin natriuretic calcium standard library of the present invention and its preparation method include, for example, the following outstanding beneficial effects:
  • the average molecular weight of the finished product of the low molecular weight heparin natriuret calcium standard library prepared by the method of the invention is 3800-5000 Daltons, and the molecular weight distribution is: the ratio of molecular weight less than 2000 is ⁇ 15.0%; the ratio of molecular weight is from 2000 to 8000 is 75.0% to 95.0%; the ratio of molecular weight is from 2000 to 4000 is from 35.0% to 55.0%, and the activity of anti-FXa factor is from 100 IU/mg to 130 IU/mg, and the ratio of anti-FXa/FIIa is ⁇ 15.0%.
  • Figure 1 is a process flow diagram of the process of the present invention.
  • a preparation method of a low molecular weight heparin nadropar calcium standard library comprises the following steps:
  • enzymatic hydrolysis adding 10 to 12 times the mass of water to the crude heparin sodium to dissolve, dissolve the first liquid solution, adjust the pH of the first liquid to 8.0 to 8.5, the temperature to 35 to 40 ° C, and add papain And ribonuclease II, enzymatic hydrolysis reaction for 8 to 10 hours, centrifugation to precipitate, the supernatant is filtered, and the first filtrate is collected;
  • Oxidative decolorization control the temperature of the first filtrate to 30-40 ° C, adjust the pH of the first filtrate to 9-11, add 0.3% to 0.5% of the first filtrate volume of hydrogen peroxide, and stir the reaction for 6-8 hours.
  • ultraviolet irradiation adding 8 to 10 times the mass of water to the second precipitate, stirring, dissolving, and irradiating with ultraviolet rays of a wavelength of 254 nm for 20 to 30 minutes at room temperature to obtain a fifth liquid;
  • oxidation adjusting the pH of the fifth liquid to 9 to 10, adding 0.8% to 1.0% of the volume of hydrogen peroxide, adjusting the temperature of the fifth liquid to 20 to 30 ° C, stirring the reaction for 6 to 8 hours; After oxidation, alcohol precipitation, static precipitation for 8-10 hours, the supernatant is removed to obtain a third precipitate, and 9-10 times of mass of water is added to the third precipitate to dissolve;
  • Nanofiltration After the reaction is completed, the replacement liquid is filtered and added to the nanofiltration machine to start circulating nanofiltration. When the volume concentration of the nanofiltration retentate reaches 20% to 25%, the second retentate is released, and the second retentate volume is added. ⁇ 3 times the amount of 95% ethanol, static precipitation for 12 to 15 hours to obtain a calcium salt precipitate; in the circulating nanofiltration process can add an appropriate amount of purified water;
  • the amount of papain in step a is from 0.5% to 0.7% by weight of crude heparin sodium, and the amount of ribonuclease II is from 0.3% to 0.5% by weight of crude heparin sodium.
  • step d sodium chloride is added to the first retentate, stirred and dissolved, and then 0.8 to 1.0 volume of 95% ethanol is added, and the precipitate is precipitated for 10 to 12 hours to separate the intermediate precipitate. Then, the intermediate precipitate is dissolved in water, and 1 to 1.2 times by volume of 95% ethanol is added to the solution, and the precipitate is precipitated for 6 to 8 hours, and the first precipitate is separated, and the first precipitate is vacuum dried and weighed; sodium chloride and The mass ratio of the first retentate is from 1.0% to 1.2%.
  • step h The specific process of alcohol precipitation in step h is: adjusting the pH value of the replacement solution to 6-8, adding sodium chloride and 2 to 3 volumes of 95% ethanol to the replacement liquid; the mass to volume ratio of sodium chloride to the replacement liquid is 0.8% to 1.0%.
  • the nitrite in step e is calcium nitrite.
  • a preparation method of a low molecular weight heparin nadropar calcium standard library comprising the following steps:
  • the concentration of glacial acetic acid is 1.8 to 2.0%; the amount of glacial acetic acid added is 7 to 8 times the mass of the first precipitate; and the amount of nitrite is 1.5% to 2.1% by weight of the first precipitate.
  • the method further comprises: oxidative decolorization of the first filtrate: controlling the temperature of the first filtrate to 30-40 ° C, adjusting the pH of the first filtrate to 9-11, adding 0.3% to 0.5 The first filtrate volume of hydrogen peroxide is stirred for 6-8 hours to obtain a purified liquid.
  • the first filtrate is used to: adjust the pH of the purified liquid to 6-8, and adopt an ultrafiltration membrane with a molecular weight cut off of 3,000-8,000 Da to carry out tangential flow circulation ultrafiltration removal of the purified liquid, and cycle ultrafiltration 5 ⁇ 8 hours, collecting first The retentate; the first retentate is removed by alcohol precipitation to obtain a first precipitate, and the first precipitate is dried and weighed.
  • the step of reducing the degradation liquid is further included: adjusting the pH value of the degradation solution to 9.0 to 10.0, adding sodium borohydride, stirring the reaction for 10 to 16 hours, adjusting the pH to 6-8, and then adding chlorine.
  • Sodium is dissolved, dissolved by stirring, and ethanol is added to the filtrate after filtration. After stirring for 20 to 30 minutes, it is allowed to stand for 8 to 12 hours; the supernatant is removed to obtain a second precipitate.
  • the method further comprises: after the second precipitate is subjected to ultraviolet irradiation, oxidation and calcium replacement, the replacement liquid is obtained, and the replacement liquid is filtered and subjected to cyclic nanofiltration, and the volume concentration of the nanofiltration retentate is filtered.
  • the ratio reaches 20% to 25%, the second retentate is released, and the second retentate is added in an amount of 2 to 3 times the amount of 95% ethanol, and the precipitate is allowed to stand for 12 to 15 hours to obtain a calcium salt precipitate.
  • the method further comprises: adding 10 to 12 times of mass of water to the calcium salt precipitate for dissolution, and completely dissolving, first filtering with a 0.2-0.3 um filter, and then using 1000 to 3000 Da.
  • Membrane circulation ultrafiltration to control molecular weight collecting the third retentate; the third retentate is filtered through a 0.1-0.2 ⁇ m decontamination membrane, collecting the third filtrate, and adjusting the pH of the third filtrate to 6.5-7.2; Perform lyophilization.
  • a low molecular weight heparin nadropar calcium standard library prepared by the preparation method of the above low molecular weight heparin nadropar calcium standard library.
  • the low molecular weight heparin nadropar calcium standard library has an average molecular weight of 3800 to 5000 Daltons.
  • the molecular weight distribution of the low molecular weight heparin nadropar calcium standard library is: the ratio of molecular weight less than 2000 is ⁇ 15.0%; the ratio of molecular weight is from 2000 to 8000 is 75.0%-95.0%; the ratio of molecular weight is from 2000 to 4000 is 35.0%-55.0. %.
  • the low molecular weight heparin nadropar calcium standard library has an anti-FXa factor activity of 100 IU/mg to 130 IU/mg, a pH of 6.5 to 7.2, and an anti-FXa/FIIa ratio of 2.7 to 4.0.
  • the low molecular weight heparin nadropar calcium is high in quality and high in quality, and can be used as a standard to establish a standard product library.
  • This embodiment provides a method for preparing a low molecular weight heparin nadropar calcium standard library, which comprises the following specific steps:
  • Enzymatic hydrolysis take 20 mg of crude heparin sodium, add 200 mg of water to dissolve it, dissolve the first liquid solution, adjust the pH of the first liquid to 8.2 with 20% (w/v) calcium hydroxide solution, adjust the first a solution temperature to 37 ° C, respectively, add papain 0.14mg, ribonuclease II 0.08mg, stirred reaction for 10 hours, centrifugation to precipitate, the supernatant was filtered with a plate and frame filter, the first filtrate was collected 200 ⁇ l;
  • step b Ultrafiltration removal: The purified liquid obtained in step b is adjusted to pH 7.2 with 4 mol/L hydrochloric acid, then subjected to tangential flow ultrafiltration, and an ultrafiltration membrane with a molecular weight cut off of 8,000 Da is selected for cyclic ultrafiltration 6 In hours, 120 ⁇ l of the first retentate was collected;
  • step d) alcohol precipitation removal adding 1.2 mg of sodium chloride to the first retentate obtained in step c), stirring and dissolving, then adding 120 ⁇ l of 95% (v / v) ethanol, precipitation for 12 hours, separation of the intermediate precipitate;
  • the intermediate precipitate was dissolved in 160 ⁇ l of water, and 192 ⁇ l of 95% (v/v) ethanol was added to the solution to precipitate for 8 hours, and the first precipitate was isolated, and the first precipitate was vacuum dried and weighed 18 mg;
  • step d Degradation: adding 126 mg of 2% (v/v) glacial acetic acid to the first precipitate obtained in step d), dissolving the second liquid solution, and adjusting the pH of the second liquid solution with 4 mol/L hydrochloric acid. To 3.1, when the temperature is adjusted to 20 ° C, 0.32 mg of calcium nitrite is added, and the reaction is stirred for 25 minutes to obtain a degradation liquid;
  • the pH of the third liquid was adjusted to 3.6 with 4 mol/L hydrochloric acid, and after stirring for 20 minutes, the fourth liquid was obtained, and the pH of the fourth liquid was adjusted with 20% (w/v) sodium hydroxide solution until 6.8, then add 1.26 mg of sodium chloride, stir to dissolve, and then filter with a micropore filter to obtain a second filtrate, and add 260 ⁇ l of 95% (v/v) ethanol to the second filtrate, stir for 25 minutes, and then let stand 10 Hour; go to the supernatant to obtain a second sediment;
  • step f ultraviolet irradiation: adding 140 mg of water to the second precipitate in step f), stirring and dissolving, and irradiating with ultraviolet rays of a wavelength of 254 nm for 25 minutes at room temperature to obtain a fifth liquid;
  • oxidation adjust the pH of the fifth liquid to 9.4 with 20% (w / v) calcium hydroxide solution, add 1.1 ⁇ l of hydrogen peroxide, adjust the temperature of the fifth liquid to 25 ° C, stir the reaction for 6 hours; After the oxidation was completed, the pH of the solution was adjusted to 7.2 with 4 mol/L hydrochloric acid, and 1.12 mg of sodium chloride and 360 ⁇ l of 95% (v/v) ethanol were added thereto, and after stirring for 20 minutes, the precipitate was allowed to stand for 10 hours, and the supernatant was removed. a third precipitate is obtained, and 160 mg of water is added to the third precipitate for dissolution;
  • Nanofiltration the reaction is completed, the filter plate is filtered, and the replacement liquid is added to the nanofiltration machine to start circulating nanofiltration, and an appropriate amount of water is added in the middle until the volume concentration of the nanofiltration retentate reaches 25%, and the second retentate is released. Adding 320 ⁇ l of 95% (v/v) ethanol, and then allowing to stand for 13 hours to prepare a calcium salt precipitate;
  • step j) Ultrafiltration purification: adding 156 mg of water to the calcium salt precipitate obtained in step j) for dissolution, completely dissolving, then filtering with a 0.22 um filter, then controlling the molecular weight with a 1000 Da membrane circulation ultrafiltration to collect the third interception
  • the third retentate is filtered through a 0.1 ⁇ m sterile membrane to obtain a third filtrate, and the pH of the third filtrate is adjusted to 6.8 with a 1 mol/L hydrochloric acid solution;
  • This embodiment provides a method for preparing a low molecular weight heparin nadropar calcium standard library, which comprises the following specific steps:
  • Enzymatic hydrolysis take 20 mg of crude heparin sodium, add 220 mg of water to dissolve, dissolve the first liquid solution, adjust the pH of the first liquid to 8.3 with 20% (w/v) calcium hydroxide solution, adjust the first a stock solution temperature to 38 ° C, respectively, add papain 0.12mg, ribonuclease II 0.1mg, stirred reaction for 10 hours, centrifuge to precipitate, the supernatant was filtered with a plate and frame filter, the first filtrate was collected 220 ⁇ l;
  • step b Ultrafiltration removal: The purified liquid obtained in step b is adjusted to pH 6.8 with 4 mol/L hydrochloric acid, and then subjected to tangential flow ultrafiltration, and an ultrafiltration membrane having a molecular weight cut off of 5,000 Da is selected for cyclic ultrafiltration for 6 hours. , collecting the first retentate 140 ⁇ l;
  • step d) alcohol precipitation and impurity removal adding 1.4 mg of sodium chloride to the first retentate obtained in step c), stirring and dissolving, then adding 140 ⁇ l of 95% (v / v) ethanol, precipitation for 11 hours, separation of the intermediate precipitate;
  • the intermediate precipitate was dissolved in 150 ⁇ l of water, and 180 ⁇ l of 95% (v/v) ethanol was added to the solution to precipitate for 8 hours, and the first precipitate was isolated, and the first precipitate was vacuum dried and weighed 17.9 mg;
  • step d) Degradation: 142 mg of 2% (v/v) glacial acetic acid was added to the first precipitate obtained in step d) to dissolve, and the second liquid solution was dissolved, and the pH of the second liquid was adjusted with 4 mol/L hydrochloric acid. To 3.1, when the temperature is adjusted to 21 ° C, 0.33 mg of calcium nitrite is added, and the reaction is stirred for 25 minutes to obtain a degradation liquid;
  • the pH of the third liquid was adjusted to 3.4 with 4 mol/L hydrochloric acid, and after stirring for 25 minutes, the fourth liquid was obtained, and the pH of the fourth liquid was adjusted with 20% (w/v) sodium hydroxide solution until 7.2, then add 1.4 mg of sodium chloride, stir to dissolve, and then filter with a micropore filter to obtain a second filtrate, and add 340 ⁇ l of 95% (v/v) ethanol to the second filtrate, stir for 20 min, and then let stand 10 Hour; go to the supernatant to obtain a second sediment;
  • step f ultraviolet irradiation: adding 152 mg of water to the second precipitate in step f), stirring and dissolving, and irradiating with ultraviolet rays of a wavelength of 254 nm for 25 minutes at room temperature to obtain a fifth liquid;
  • oxidation adjust the pH of the fifth liquid to 9.6 with 20% (w / v) calcium hydroxide solution, add 1.2 ⁇ l of hydrogen peroxide, adjust the temperature of the fifth liquid to 26 ° C, stir the reaction for 6 hours; After the oxidation was completed, the pH of the solution was adjusted to 7.0 with 4 mol/L hydrochloric acid, 1.2 mg of sodium chloride and 400 ⁇ l of 95% (v/v) ethanol were added thereto, and after stirring for 20 minutes, the precipitate was allowed to stand for 10 hours, and the supernatant was removed. a third precipitate is obtained, and 160 mg of water is added to the third precipitate for dissolution;
  • Nanofiltration the reaction is completed, the filter plate is filtered, and the replacement liquid is added to the nanofiltration machine to start circulating nanofiltration, and an appropriate amount of water is added in the middle until the volume concentration of the nanofiltration retentate reaches 25%, and the second retentate is released. Adding 400 ⁇ l of 95% (v/v) ethanol, and then allowing to stand for 12 hours to prepare a calcium salt precipitate;
  • This embodiment provides a method for preparing a low molecular weight heparin nadropar calcium standard library, which comprises the following specific steps:
  • Enzymatic hydrolysis take 20 mg of crude heparin sodium, add 240 mg of water to dissolve it, dissolve the first liquid solution, adjust the pH of the first liquid to 8.0 with 20% (w/v) calcium hydroxide solution, adjust the first a solution temperature to 35 ° C, respectively, add papain 0.1 mg, ribonuclease II 0.06 mg, stir the reaction for 8 hours, centrifuge to precipitate, the supernatant was filtered with a plate and frame filter, the first filtrate was collected 200 ⁇ l;
  • step b Ultrafiltration removal: The purified liquid obtained in step b is adjusted to pH 6 with 4 mol/L hydrochloric acid, and then subjected to tangential flow ultrafiltration, and an ultrafiltration membrane having a molecular weight cut off of 3,000 Da is selected for cyclic ultrafiltration 8 In hours, 120 ⁇ l of the first retentate was collected;
  • step d Degradation: adding 144 mg of 2% (v/v) glacial acetic acid to the first precipitate obtained in step d), dissolving to obtain a second liquid solution, and adjusting the pH of the second liquid solution with 4 mol/L hydrochloric acid To 3.0, when the temperature is adjusted to 18 ° C, 0.38 mg of calcium nitrite is added, and the reaction is stirred for 25 minutes to obtain a degradation liquid;
  • 20% (w / v) sodium hydroxide solution adjust the pH of the fourth liquid to 6, then add 1.26mg of sodium chloride, stir to dissolve, and then filter with a microporous filter to obtain a second filtrate, and to the second 260 ⁇ l of 95% (v/v) ethanol was added to the filtrate, and after stirring for 30 minutes, it was allowed to stand for 8 hours; the supernatant was removed to obtain a second precipitate;
  • step f ultraviolet irradiation: adding 140 mg of water to the second precipitate in step f), stirring, dissolving, and irradiating with ultraviolet rays of a wavelength of 254 nm for 20 minutes at room temperature to obtain a fifth liquid;
  • oxidation adjust the pH of the fifth liquid to 9.0 with 20% (w / v) calcium hydroxide solution, add 1.1 ⁇ l of hydrogen peroxide, adjust the temperature of the fifth liquid to 20 ° C, stir the reaction for 6 hours; After the oxidation was completed, the pH of the solution was adjusted to 7.2 with 4 mol/L hydrochloric acid, and 1.12 mg of sodium chloride and 360 ⁇ l of 95% (v/v) ethanol were added thereto, and after stirring for 20 minutes, the precipitate was allowed to stand for 8 hours, and the supernatant was removed. a third precipitate is obtained, and 160 mg of water is added to the third precipitate for dissolution;
  • Nanofiltration the reaction is completed, the filter plate is filtered, and the replacement liquid is added to the nanofiltration machine to start circulating nanofiltration, and an appropriate amount of water is added in the middle until the volume concentration of the nanofiltration retentate reaches 20%, and the second retentate is released. Adding 320 ⁇ l of 95% (v/v) ethanol, and then allowing to stand for 13 hours to prepare a calcium salt precipitate;
  • This embodiment provides a method for preparing a low molecular weight heparin nadropar calcium standard library, which comprises the following specific steps:
  • Enzymatic hydrolysis take 20 mg of crude heparin sodium, add 220 mg of water to dissolve it, dissolve the first liquid solution, adjust the pH of the first liquid to 8.5 with 20% (w/v) calcium hydroxide solution, adjust the first One temperature of the solution was adjusted to 40 ° C, 0.13 mg of papain and 0.1 mg of ribonuclease II were added, respectively, and the reaction was stirred for 9 hours, centrifuged to precipitate, and the supernatant was filtered through a plate and frame filter to collect 220 ⁇ l of the first filtrate. ;
  • step b Ultrafiltration removal: the purified liquid obtained in step b is adjusted to pH 8 with 4 mol/L hydrochloric acid, and then subjected to tangential flow ultrafiltration, and an ultrafiltration membrane having a molecular weight cut off of 6,000 Da is selected for cyclic ultrafiltration for 5 hours. , collecting the first retentate 140 ⁇ l;
  • step d Degradation: adding 143.2 mg of 2% (v/v) glacial acetic acid to the precipitate obtained in step d), dissolving to obtain a second liquid solution, and adjusting the pH of the second liquid to 4 mol/L hydrochloric acid to 3.2, when the temperature is adjusted to 22 ° C, 0.38 mg of calcium nitrite is added, and the reaction is stirred for 25 minutes to obtain a degradation liquid;
  • the pH of the third liquid was adjusted to 4.0 with 4 mol/L hydrochloric acid, and after stirring for 30 minutes, the fourth liquid was obtained, and the pH of the fourth liquid was adjusted with 20% (w/v) sodium hydroxide solution until 8, then added 1.4 mg of sodium chloride, stirred to dissolve, and then filtered through a micropore filter to obtain a second filtrate, and added 340 ⁇ l of 95% (v / v) ethanol to the second filtrate, stirred for 20 min, and then allowed to stand 12 Hour; go to the supernatant to obtain a second sediment;
  • step f) ultraviolet irradiation: the supernatant in step f) is de-cleared, added with 152 mg of water, stirred to dissolve, and irradiated with ultraviolet light of a wavelength of 254 nm for 30 minutes at room temperature to obtain a fifth liquid;
  • oxidation adjust the pH of the fifth liquid to 10.0 with 20% (w / v) calcium hydroxide solution, add 1.2 ⁇ l of hydrogen peroxide, adjust the temperature of the fifth liquid to 30 ° C, stir the reaction for 6 hours; After the oxidation was completed, the pH of the solution was adjusted to 7.0 with 4 mol/L hydrochloric acid, 1.2 mg of sodium chloride and 400 ⁇ l of 95% (v/v) ethanol were added thereto, and after stirring for 20 minutes, the precipitate was statically precipitated for 9 hours, and the supernatant was removed. a third precipitate is obtained, and 160 mg of water is added to the third precipitate for dissolution;
  • Nanofiltration the reaction is completed, the filter plate is filtered, and the replacement liquid is added to the nanofiltration machine to start circulating nanofiltration, and an appropriate amount of water is added in the middle until the volume concentration of the nanofiltration retentate reaches 23%, and the second retentate is released. Adding 400 ⁇ l of 95% (v/v) ethanol, and then allowing to stand for 12 hours to prepare a calcium salt precipitate;
  • step j) Ultrafiltration refining: adding 1600 mg of water to the calcium salt precipitate obtained in step j) for dissolution, completely dissolving, then filtering with a 0.22 um filter, then controlling the molecular weight with 3000 Da membrane circulation ultrafiltration to collect the third interception
  • the third retentate is filtered through a 0.1 ⁇ m sterile membrane to obtain a third filtrate, and the pH of the third filtrate is adjusted to 7.2 with a 1 mol/L hydrochloric acid solution;
  • the low molecular weight heparin nadroparin calcium standard library of the present invention and its preparation method include, for example, the following outstanding effects:
  • the average molecular weight of the finished product of the low molecular weight heparin natriuret calcium standard library prepared by the method of the invention is 3800-5000 Daltons, and the molecular weight distribution is: the ratio of molecular weight less than 2000 is ⁇ 15.0%; the ratio of molecular weight is from 2000 to 8000 is 75.0% to 95.0%; the ratio of molecular weight is from 2000 to 4000 is from 35.0% to 55.0%, and the activity of anti-FXa factor is from 100 IU/mg to 130 IU/mg, and the ratio of anti-FXa/FIIa is ⁇ 15.0%.

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Abstract

本发明公开了一种低分子肝素那曲肝素钙标准品库的制备方法及其产品，该方法以粗品肝素钠为原料，以酶解和改良的亚硝酸盐降解法为基础，经酶解、氧化脱色、超滤除杂、醇沉除杂、降解、还原、紫外照射、氧化、钙置换、纳滤、超滤精制、冻干等步骤制得具有特定平均分子量的低分子肝素那曲肝素钙标准品库。

Description

低分子肝素那曲肝素钙标准品库及其制备方法 技术领域
本发明涉及生物医药领域，具体地说是一种低分子肝素那曲肝素钙标准品库(Nadroparin)及其制备方法。
背景技术
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低分子肝素那曲肝素钙标准品库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。
低分子肝素那曲肝素钙是通过酸性亚硝酸钙的解聚，硼氢化钠还原和紫外修饰，经氧化、钙置换后的一种抗凝药物，是低分子肝素的一种。通过利用该低分子肝素那曲肝素钙形成标准品库，用于研究，因此需要高品质、高质量的低分子肝素那曲肝素钙。国际和国内上制备方法比较单一，均为亚硝酸盐降解，但以此为条件，产品降解不稳定，内部结构破坏较大，结构确认合格率低。目前通常的制备方法是：将外购的精品肝素钠原料溶于纯化水中，直接加入一定量的亚硝酸钠，控制pH值在2.7～2.9范围内，一定温度下搅拌反应25～35min。再经还原、转钙、过滤、浓缩等制的成品。传统方法是以外购的精品肝素为原料，难以从原料源头控制最终成品质量，降解时只选择了肝素可被亚硝酸盐降解而生产低分子，而其降解过程难以控制pH值的剧烈变化而带来对产品分子量和结构的不可控性，且产品稳定性差。转钙时只是以传统的沉淀方法进行产品中游离钙离子的去除，难以达到可控的效果，从而导致制备出来的产品纯度不够，结构不稳定等缺点，无法作为标准品，这些都是目前工艺中不能解决的问题。而且在制成的注射剂药物的过程中存在一定的风险。
发明内容
本发明的第一目的在于是针对上述现有技术的不足，提供一种低分子肝素那曲肝素钙标准品库的制备方法。该方法克服了传统工艺中的原料不可控性及产品产率低，产品稳定 性差的缺陷。以该方法生产的低分子肝素那曲肝素钙标准品库的质量高于国有标准和欧洲质量标准，且稳定性好，产品收率高。
本发明的第二目的在于提供一种低分子肝素那曲肝素钙标准品库，其由上述低分子肝素那曲肝素钙标准品库的制备方法制得，其稳定性好，生物利用度高、体内半衰期长、出血倾向小。
本发明的技术任务是按以下方式实现的：该方法采用粗品肝素钠为原料，选用木瓜蛋白酶、核糖核酸酶II等蛋白和核酸酶优化组合，对粗品肝素钠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。将经过处理的肝素钠加入具有缓冲作用的冰醋酸中完全溶解，在特定温度和酸性条件下，与亚硝酸盐如亚硝酸钙反应，引起N-硫酸基氨基葡糖残基上的脱氨作用，进而发生重排反应引起氨基葡糖和糖醛酸之间的糖苷键断裂，形成2，5-脱水甘露糖末端残基，然后还原处理获得稳定的2，5-脱水甘露醇，紫外照射后再经过钙盐置换取代结构上的钠离子，后经纳滤技术和低分子膜分离技术，得到分子量3800～5000道尔顿的低分子肝素那曲肝素钙标准品库粗品，将上述低分子肝素那曲肝素钙标准品库粗品经过氧化氢氧化脱色，乙醇沉淀、真空冷冻干燥，得到抗FXa因子活性为100IU/mg～130IU/mg、pH值为6.5～7.2、抗FXa/FIIa比值为2.7～4.0、平均分子量为3800～5000D的低分子肝素那曲肝素钙标准品库成品。
第一方面，本申请实施例提供了一种低分子肝素那曲肝素钙标准品库的制备方法，包括以下步骤：
a)酶解：向粗品肝素钠中加入10～12倍质量的水溶解，溶解得第一料液，调节第一料液的pH值至8.0～8.5，温度至35～40℃，加入木瓜蛋白酶及核糖核酸酶II，酶解反应8～10小时，离心去沉淀，上清液过滤，收集第一滤液；
b)氧化脱色：控制第一滤液温度至30～40℃，调整第一滤液pH值至9～11，加入0.3％～0.5％第一滤液体积的过氧化氢，搅拌反应6～8小时，得纯化液；
c)超滤除杂：调节纯化液的pH值至6～8，采用截留分子量为3,000～8,000Da的超滤膜对纯化液进行切向流循环超滤，循环超滤5～8小时，收集第一截留液；
d)醇沉除杂：第一截留液经醇沉除杂，得到第一沉淀物，对第一沉淀物干燥称重；
e)降解：向第一沉淀物中加入7～8倍质量的冰醋酸溶解，溶解得第二料液，调节第二料液pH值至3.0～3.2，调整第二料液温度至18～22℃，加入亚硝酸盐，搅拌反应20～30分钟，得到降解液；所述冰醋酸的体积浓度为1.8％～2.0％；所述亚硝酸盐的用量为第一沉淀物重量的1.5％～2.1％；
f)还原：降解反应完成后，调节降解液的pH值至9.0～10.0，加入第一沉淀物重量0.9％～ 1.1％的硼氢化钠，搅拌反应10～15h，得第三料液，反应完成后调节第三料液的pH值至3.0～4.0，搅拌反应15-30分钟后，得第四料液，再调节第四料液的pH值至6～8，然后加入氯化钠，搅拌溶解，微孔过滤器过滤，并向其中加入2～3倍体积的95％乙醇，搅拌20～30min后，静置8～12小时；去上清，得第二沉淀物；氯化钠与第四料液的质量体积比为0.9％～1.1％；
g)紫外照射：向第二沉淀物中加入8～10倍质量的水搅拌使其溶解，在室温下用254nm的波长的紫外线照射20～30分钟，得第五料液；
h)氧化：调节第五料液pH值至9～10，加入0.8％～1.0％料液体积的过氧化氢，调节第五料液的温度至20～30℃，搅拌反应6～8小时；氧化完毕，醇沉，静止沉淀8～10小时后，去上清，得第三沉淀物，向第三沉淀物中加入9～10倍质量的水溶解；
i)钙置换：在搅拌状态下持续加入氯化钙，20～25℃下搅拌反应10～12小时，得置换液；所述氯化钙的用量为第三沉淀物重量的8～12％；
j)纳滤：反应完成，置换液经过滤后加入纳滤机开始循环纳滤，纳滤截留液的体积浓度达到20％～25％时，放出第二截留液，加入第二截留液体积2～3倍量的95％乙醇，静置沉淀12～15小时制得钙盐沉淀物；循环纳滤过程中可补加适量纯化水；
k)超滤精制：向钙盐沉淀物加入10～12倍质量的水进行溶解，完全溶解后先用0.22um的滤膜过滤，然后用1000～3000Da的膜循环超滤控制分子量，收集第三截留液；第三截留液经0.1μm的除菌膜过滤，收集第三滤液，并调节第三滤液pH值至6.5～7.2；
l)将第三滤液进行冻干处理，得低分子肝素那曲肝素钙标准品库成品。
结合第一方面，在本发明的其他优选实施例中，步骤a中木瓜蛋白酶的用量为粗品肝素钠重量的0.5％～0.7％，核糖核酸酶II的用量为粗品肝素钠重量的0.3％～0.5％。
步骤d、步骤h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：
结合第一方面，在本发明的其他优选实施例中，步骤d中醇沉除杂时，向第一截留液中加入氯化钠，搅拌溶解后再加入0.8～1.0倍体积的95％乙醇，沉淀10～12小时，分离得中间沉淀物；再将中间沉淀物加水溶解，向溶液中加入l～1.2倍体积的95％乙醇，沉淀6～8小时，分离得第一沉淀物，第一沉淀物真空干燥并称重；氯化钠与第一截留液的质量体积比为1.0％～1.2％。
结合第一方面，在本发明的其他优选实施例中，步骤h中醇沉的具体过程为：调节置换液pH值至6～8，向置换液中加入氯化钠及2～3倍体积的95％乙醇；氯化钠与置换液的质量体积比为0.8％～1.0％。
结合第一方面，在本发明的其他优选实施例中，步骤e中亚硝酸盐为亚硝酸钙。
第二方面，本申请实施例提供了一种低分子肝素那曲肝素钙标准品库的制备方法，其包括以下步骤：
向粗品肝素钠的水溶液中加入木瓜蛋白酶及核糖核酸酶II，酶解后离心去沉淀，收集第一滤液；
第一滤液经多次除杂后，得第一沉淀物；
向第一沉淀物中加入冰醋酸溶解，调节pH至3.0～3.2，调节温度至18～22℃，接着加入亚硝酸盐进行降解，搅拌反应20～30分钟，得降解液。
结合第二方面，在本发明的其他优选实施例中，冰醋酸的浓度为1.8～2.0％；冰醋酸的加入量为第一沉淀物的质量的7～8倍；亚硝酸盐的用量为第一沉淀物重量的1.5％～2.1％。
结合第二方面，在本发明的其他优选实施例中，在对第一滤液进行多次除杂之前，还包括对第一滤液进行氧化脱色：控制第一滤液温度至30～40℃，调整第一滤液pH值至9～11，加入0.3％～0.5％第一滤液体积的过氧化氢，搅拌反应6～8小时，得纯化液。
结合第二方面，在本发明的其他优选实施例中，多次除杂第一滤液包括：调节纯化液pH值至6～8，采用截留分子量为3,000～8,000Da的超滤膜对纯化液进行切向流循环超滤除杂，循环超滤5～8小时，收集第一截留液；第一截留液经醇沉除杂，得到第一沉淀物，对第一沉淀物干燥称重。
结合第二方面，在本发明的其他优选实施例中，在制得降解液之后，还包括还原降解液步骤：调节降解液的pH值至9.0～10.0，加入硼氢化钠，搅拌反应10～16h，调节pH值至6～8，然后加入氯化钠，搅拌溶解，过滤后向滤液中加入乙醇，搅拌20～30min后，静置8～12小时；去上清，得第二沉淀物。
结合第二方面，在本发明的其他优选实施例中，在还原降解液步骤之后，还包括：将第二沉淀物经紫外照射、氧化和钙置换后，得置换液，将置换液经过滤后进行循环纳滤，纳滤截留液的体积浓度达到20％～25％时，放出第二截留液，加入第二截留液体积2～3倍量的95％乙醇，静置沉淀12～15小时制得钙盐沉淀物。
结合第二方面，在本发明的其他优选实施例中，在制得钙盐沉淀物之后，还包括：向钙盐沉淀物加入10～12倍质量的水进行溶解，完全溶解后先用0.2～0.3um的滤膜过滤，然后用1000～3000Da的膜循环超滤控制分子量，收集第三截留液；第三截留液经0.1～0.2μm的除菌膜过滤，收集第三滤液，并调节第三滤液pH值至6.5～7.2；将第三滤液进行冻干处理。
第三方面，本申请实施例还提供了一种低分子肝素那曲肝素钙标准品库，其是由上述低分子肝素那曲肝素钙标准品库的制备方法制得。
结合第三方面，在本发明的其他优选实施例中，低分子肝素那曲肝素钙标准品库的平均分子量为3800～5000道尔顿。
结合第三方面，在本发明的其他优选实施例中，低分子肝素那曲肝素钙标准品库的分子量分布为：分子量小于2000的比率为≤15.0％；分子量于2000～8000的比率为75.0％～95.0％；分子量在2000～4000的比率为35.0％～55.0％。
结合第三方面，在本发明的其他优选实施例中，低分子肝素那曲肝素钙标准品库的抗FXa因子活性为100IU/mg～130IU/mg、pH值为6.5～7.2、抗FXa/FIIa比值为2.7～4.0。
与现有技术相比，本发明的低分子肝素那曲肝素钙标准品库及其制备方法例如包括以下突出的有益效果：
(一)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，对粗品肝素钠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；
(二)进行亚硝酸盐降解前，将经过处理的肝素钠加入具有缓冲作用的冰醋酸，克服了传统工艺与亚硝酸盐反应时的pH变化剧烈而引起的产品分子量剧烈变化，生产产品结构不合格，产品稳定性差等缺陷，增加产品的稳定性；
(三)在一定温度和酸性条件下，与亚硝酸钙发生降解反应。采用亚硝酸钙降解，会有部分钙离子置换出产品中的钠离子，这样可以适当减少后期钙置换时氯化钙的投入，及其钙置换的反应时间，降低了成本投入和缩短了制备周期；
(四)将先进的纳滤技术应用于产品中游离钙离子的去除，改变了落后的溶媒分级沉淀去除法，产品收率大幅提高；
(五)由于本实施例中的目的在于生产标准品，主要用于研究而非生产，因此该工艺的成本投入以及收率等因素不作为本实施例的主要考量对象，最终成品的品质和质量作为最主要的考量对象，本实施例中，通过本发明方法制备得到的低分子肝素那曲肝素钙标准品库成品的平均分子量为3800～5000道尔顿，分子量分布为：分子量小于2000的比率为≤15.0％；分子量于2000～8000的比率为75.0％～95.0％；分子量在2000～4000的比率为35.0％～55.0％，抗FXa因子活性为100IU/mg～130IU/mg，抗FXa/FIIa比值为2.7～4.0，pH值为6.5～7.2；5％的产品水溶液澄清透明；乙醇含量≤0.8％；钙含量(干品)9.7％～11.3％，产品质量超越国内标准和欧洲药典标准，生物利用度高、体内半衰期长、出血倾 向小；该低分子肝素那曲肝素钙的成品品质高，质量高，能够作为标准品以建立标准品库；
(六)操作简单，制得的成品的品质高、质量高。
附图说明
附图1是本发明方法的工艺流程图。
具体实施方式
参照说明书附图以具体实施例对本发明的低分子肝素那曲肝素钙标准品库及其制备方法作以下详细地说明。
一种低分子肝素那曲肝素钙标准品库的制备方法，包括以下步骤：
a)酶解：向粗品肝素钠中加入10～12倍质量的水溶解，溶解得第一料液，调节第一料液的pH值至8.0～8.5，温度至35～40℃，加入木瓜蛋白酶及核糖核酸酶II，酶解反应8～10小时，离心去沉淀，上清液过滤，收集第一滤液；
b)氧化脱色：控制第一滤液温度至30～40℃，调整第一滤液pH值至9～11，加入0.3％～0.5％第一滤液体积的过氧化氢，搅拌反应6～8小时，得纯化液；
c)超滤除杂：调节纯化液的pH值至6～8，采用截留分子量为3,000～8,000Da的超滤膜对纯化液进行切向流循环超滤，循环超滤5～8小时，收集第一截留液；
d)醇沉除杂：第一截留液经醇沉除杂，得到第一沉淀物，对第一沉淀物干燥称重；
e)降解：向第一沉淀物中加入7～8倍质量的冰醋酸溶解，溶解得第二料液，调节第二料液pH值至3.0～3.2，调整第二料液温度至18～22℃，加入亚硝酸盐，搅拌反应20～30分钟，得到降解液；冰醋酸的体积浓度为1.8％～2.0％；亚硝酸盐的用量为第一沉淀物重量的1.5％～2.1％；
f)还原：降解反应完成后，调节降解液的pH值至9.0～10.0，加入第一沉淀物重量0.9％～1.1％的硼氢化钠，搅拌反应10～15h，得第三料液，反应完成后调节第三料液的pH值至3.0～4.0，搅拌反应15-30分钟后，得第四料液，再调节第四料液的pH值至6～8，然后加入氯化钠，搅拌溶解，微孔过滤器过滤，并向其中加入2～3倍体积的95％乙醇，搅拌20～30min后，静置8～12小时；去上清，得第二沉淀物；氯化钠与第四料液的质量体积比为0.9％～1.1％；
g)紫外照射：向第二沉淀物中加入8～10倍质量的水搅拌使其溶解，在室温下用254nm的波长的紫外线照射20～30分钟，得第五料液；
h)氧化：调节第五料液pH值至9～10，加入0.8％～1.0％料液体积的过氧化氢，调节第五料液的温度至20～30℃，搅拌反应6～8小时；氧化完毕，醇沉，静止沉淀8～10小时后，去上清，得第三沉淀物，向第三沉淀物中加入9～10倍质量的水溶解；
i)钙置换：在搅拌状态下持续加入氯化钙，20～25℃下搅拌反应10～12小时，得置换液；氯化钙的用量为第三沉淀物重量的8～12％；
j)纳滤：反应完成，置换液经过滤后加入纳滤机开始循环纳滤，纳滤截留液的体积浓度达到20％～25％时，放出第二截留液，加入第二截留液体积2～3倍量的95％乙醇，静置沉淀12～15小时制得钙盐沉淀物；循环纳滤过程中可补加适量纯化水；
k)超滤精制：向钙盐沉淀物加入10～12倍质量的水进行溶解，完全溶解后先用0.22um的滤膜过滤，然后用1000～3000Da的膜循环超滤控制分子量，收集第三截留液；第三截留液经0.1μm的除菌膜过滤，收集第三滤液，并调节第三滤液pH值至6.5～7.2；
l)将第三滤液进行冻干处理，得低分子肝素那曲肝素钙标准品库成品。
优选地，步骤a中木瓜蛋白酶的用量为粗品肝素钠重量的0.5％～0.7％，核糖核酸酶II的用量为粗品肝素钠重量的0.3％～0.5％。
可选地，步骤d中醇沉除杂时，向第一截留液中加入氯化钠，搅拌溶解后再加入0.8～1.0倍体积的95％乙醇，沉淀10～12小时，分离得中间沉淀物；再将中间沉淀物加水溶解，向溶液中加入l～1.2倍体积的95％乙醇，沉淀6～8小时，分离得第一沉淀物，第一沉淀物真空干燥并称重；氯化钠与第一截留液的质量体积比为1.0％～1.2％。
步骤h中醇沉的具体过程为：调节置换液pH值至6～8，向置换液中加入氯化钠及2～3倍体积的95％乙醇；氯化钠与置换液的质量体积比为0.8％～1.0％。
优选地，步骤e中亚硝酸盐为亚硝酸钙。
一种低分子肝素那曲肝素钙标准品库的制备方法，其包括以下步骤：
向粗品肝素钠的水溶液中加入木瓜蛋白酶及核糖核酸酶II，酶解后离心去沉淀，收集第一滤液；
第一滤液经除杂后，得第一沉淀物；
向第一沉淀物中加入冰醋酸溶解，调节pH至3.0～3.2，调节温度至18～22℃，接着加入亚硝酸盐进行降解，搅拌反应20～30分钟，得降解液。
优选地，冰醋酸的浓度为1.8～2.0％；冰醋酸的加入量为第一沉淀物的质量的7～8倍；亚硝酸盐的用量为第一沉淀物重量的1.5％～2.1％。
进一步地，在对第一滤液进行除杂之前，还包括对第一滤液进行氧化脱色：控制第一滤液温度至30～40℃，调整第一滤液pH值至9～11，加入0.3％～0.5％第一滤液体积的过氧化氢，搅拌反应6～8小时，得纯化液。
优选地，除杂第一滤液包括：调节纯化液pH值至6～8，采用截留分子量为3,000～8,000Da的超滤膜对纯化液进行切向流循环超滤除杂，循环超滤5～8小时，收集第一 截留液；第一截留液经醇沉除杂，得到第一沉淀物，对第一沉淀物干燥称重。
进一步地，在制得降解液之后，还包括还原降解液步骤：调节降解液的pH值至9.0～10.0，加入硼氢化钠，搅拌反应10～16h，调节pH值至6～8，然后加入氯化钠，搅拌溶解，过滤后向滤液中加入乙醇，搅拌20～30min后，静置8～12小时；去上清，得第二沉淀物。
进一步地，在还原降解液步骤之后，还包括：将第二沉淀物经紫外照射、氧化和钙置换后，得置换液，将置换液经过滤后进行循环纳滤，纳滤截留液的体积浓度达到20％～25％时，放出第二截留液，加入第二截留液体积2～3倍量的95％乙醇，静置沉淀12～15小时制得钙盐沉淀物。
进一步地，在制得钙盐沉淀物之后，还包括：向钙盐沉淀物加入10～12倍质量的水进行溶解，完全溶解后先用0.2～0.3um的滤膜过滤，然后用1000～3000Da的膜循环超滤控制分子量，收集第三截留液；第三截留液经0.1～0.2μm的除菌膜过滤，收集第三滤液，并调节第三滤液pH值至6.5～7.2；将第三滤液进行冻干处理。
一种低分子肝素那曲肝素钙标准品库，其是由上述低分子肝素那曲肝素钙标准品库的制备方法制得。
其中，低分子肝素那曲肝素钙标准品库的平均分子量为3800～5000道尔顿。低分子肝素那曲肝素钙标准品库的分子量分布为：分子量小于2000的比率为≤15.0％；分子量于2000～8000的比率为75.0％～95.0％；分子量在2000～4000的比率为35.0％～55.0％。
低分子肝素那曲肝素钙标准品库的抗FXa因子活性为100IU/mg～130IU/mg、pH值为6.5～7.2、抗FXa/FIIa比值为2.7～4.0。
该低分子肝素那曲肝素钙的成品品质高，质量高，能够作为标准品以建立标准品库。
制备实施例一：
本实施例提供了一种低分子肝素那曲肝素钙标准品库的制备方法，其包括以下具体步骤：
a)酶解：取粗品肝素钠20mg，并向其中加入200mg水溶解，溶解得第一料液，用20％(w/v)氢氧化钙溶液调节第一料液pH值至8.2，调节第一料液温度至37℃，分别加入木瓜蛋白酶0.14mg、核糖核酸酶II 0.08mg，搅拌反应10小时，离心去沉淀，将上清液用板框过滤器过滤，收集得第一滤液200μl；
b)氧化：控制步骤a)所得第一滤液温度至37℃，用20％(w/v)氢氧化钙溶液调整第一滤液pH值至10.1，加入0.6μl过氧化氢，室温搅拌反应8小时，得纯化液；
c)超滤除杂：将步骤b中所获得的纯化液用4mol/L的盐酸调节为pH7.2后进行切向流超滤，选择截留分子量为8,000Da的超滤膜进行循环超滤6小时，收集得第一截留液120μl；
d)醇沉除杂：向步骤c)所得的第一截留液中加入1.2mg氯化钠，搅拌溶解后再加入120μl 95％(v/v)乙醇，沉淀12小时，分离得中间沉淀物；中间沉淀物加160μl水溶解，并向溶液中加入192μl 95％(v/v)乙醇，沉淀8小时，分离得第一沉淀物，第一沉淀物真空干燥，重18mg；
e)降解：向步骤d)中所得的第一沉淀物中加入126mg 2％(v/v)的冰醋酸溶解，溶解得第二料液，用4mol/L的盐酸调节第二料液pH值至3.1，调整温度至20℃时，加入0.32mg亚硝酸钙，搅拌反应25分钟，得到降解液；
f)还原：反应完成后，迅速用20％(w/v)氢氧化钙溶液调节降解液的pH值至9.4，加入0.2mg硼氢化钠，搅拌反应12h，反应完成后得第三料液，用4mol/L的盐酸调节第三料液的pH值至3.6，搅拌反应20分钟后，得第四料液，用20％(w/v)氢氧化钠溶液调节第四料液的pH值至6.8，然后加入1.26mg氯化钠，搅拌溶解，然后用微孔过滤器过滤，得第二滤液，并向第二滤液中加入260μl 95％(v/v)乙醇，搅拌25min后，静置10小时；去上清，得第二沉淀物；
g)紫外照射：向步骤f)中的第二沉淀物加入140mg水搅拌使其溶解，在室温下用254nm的波长的紫外线照射25分钟，得第五料液；
h)氧化：用20％(w/v)氢氧化钙溶液调节第五料液pH值至9.4，加入1.1μl的过氧化氢，调节第五料液的温度至25℃，搅拌反应6小时；氧化完毕后，用4mol/L的盐酸调节料液pH值至7.2，向其中加入1.12mg氯化钠及360μl 95％(v/v)乙醇，搅拌20分钟以后，静止沉淀10小时，去上清，得第三沉淀物，向第三沉淀物中加入160mg水进行溶解；
i)钙置换：在搅拌状态下持续加入1.28mg氯化钙，20℃下搅拌反应12小时，得置换液；
j)纳滤：反应完成，滤板过滤，将置换液加入纳滤机开始循环纳滤，中间补加适量的水，直到纳滤截留液的体积浓度达到25％时，放出第二截留液，加入320μl 95％(v/v)乙醇，然后静置沉淀13小时制得钙盐沉淀物；
k)超滤精制：向步骤j)中所得的钙盐沉淀物加入156mg水进行溶解，完全溶解后先用0.22um的滤膜过滤，然后用1000Da的膜循环超滤控制分子量，收集第三截留液；将第三截留液经0.1μm的除菌膜过滤后，得第三滤液，再用1mol/L的盐酸溶液调节第三滤液pH值至6.8；
L)将第三滤液转移至真空冷冻干燥箱中进行冻干处理，得低分子肝素那曲肝素钙标准品库成品15.4mg。
制备实施例二：
本实施例提供了一种低分子肝素那曲肝素钙标准品库的制备方法，其包括以下具体步骤：
a)酶解：取粗品肝素钠20mg，并向其中加入220mg水溶解，溶解得第一料液，用20％(w/v)氢氧化钙溶液调节第一料液pH值至8.3，调节第一料液温度至38℃，分别加入木瓜蛋白酶0.12mg、核糖核酸酶II 0.1mg，搅拌反应10小时，离心去沉淀，将上清液用板框过滤器过滤，收集得第一滤液220μl；
b)氧化：控制步骤a)所得第一滤液温度至39℃，用20％(w/v)氢氧化钙溶液调整第一滤液pH值至9.5，加入0.7μl过氧化氢，室温搅拌反应7小时，得纯化液；
c)超滤除杂：将步骤b中所获得的纯化液用4mol/L的盐酸调节pH为6.8后进行切向流超滤，选择截留分子量为5,000Da的超滤膜进行循环超滤6小时，收集得第一截留液140μl；
d)醇沉除杂：向步骤c)所得的第一截留液中加入1.4mg氯化钠，搅拌溶解后再加入140μl 95％(v/v)乙醇，沉淀11小时，分离得中间沉淀物；中间沉淀物加150μl水溶解，并向溶液中加入180μl 95％(v/v)乙醇，沉淀8小时，分离得第一沉淀物，第一沉淀物真空干燥，重17.9mg；
e)降解：向步骤d)中所得的第一沉淀物中加入142mg 2％(v/v)的冰醋酸溶解，溶解得第二料液，用4mol/L的盐酸调节第二料液pH值至3.1，调整温度至21℃时，加入0.33mg亚硝酸钙，搅拌反应25分钟，得到降解液；
f)还原：反应完成后，迅速用20％(w/v)氢氧化钙溶液调节降解液的pH值至9.5，加入0.18mg硼氢化钠，搅拌反应12h，反应完成后得第三料液，用4mol/L的盐酸调节第三料液的pH值至3.4，搅拌反应25分钟后，得第四料液，用20％(w/v)氢氧化钠溶液调节第四料液的pH值至7.2，然后加入1.4mg氯化钠，搅拌溶解，然后用微孔过滤器过滤，得第二滤液，并向第二滤液中加入340μl 95％(v/v)乙醇，搅拌20min后，静置10小时；去上清，得第二沉淀物；
g)紫外照射：向步骤f)中的第二沉淀物加入152mg水搅拌使其溶解，在室温下用254nm的波长的紫外线照射25分钟，得第五料液；
h)氧化：用20％(w/v)氢氧化钙溶液调节第五料液pH值至9.6，加入1.2μl的过氧化氢，调节第五料液的温度至26℃，搅拌反应6小时；氧化完毕后，用4mol/L的盐酸调节料液pH值至7.0，向其中加入1.2mg氯化钠及400μl 95％(v/v)乙醇，搅拌20分钟以后，静止沉淀10小时，去上清，得第三沉淀物，向第三沉淀物中加入160mg水进行溶解；
i)钙置换：在搅拌状态下持续加入1.28mg氯化钙，21℃下搅拌反应12小时，得置换液；
j)纳滤：反应完成，滤板过滤，将置换液加入纳滤机开始循环纳滤，中间补加适量的水，直到纳滤截留液的体积浓度达到25％时，放出第二截留液，加入400μl 95％(v/v)乙醇，然后静置沉淀12小时制得钙盐沉淀物；
k)超滤精制：向步骤j)中所得的钙盐沉淀物加入1600mg水进行溶解，完全溶解后先用0.22um的滤膜过滤，然后用1000Da的膜循环超滤控制分子量，收集第三截留液；将第三截留液经0.1μm的除菌膜过滤后，得第三滤液，再用1mol/L的盐酸溶液调节滤液pH值至6.7；
L)将第三滤液转移至真空冷冻干燥箱中进行冻干处理，得低分子肝素那曲肝素钙标准品库成品15.3mg。
制备实施例三：
本实施例提供了一种低分子肝素那曲肝素钙标准品库的制备方法，其包括以下具体步骤：
a)酶解：取粗品肝素钠20mg，并向其中加入240mg水溶解，溶解得第一料液，用20％(w/v)氢氧化钙溶液调节第一料液pH值至8.0，调节第一料液温度至35℃，分别加入木瓜蛋白酶0.1mg、核糖核酸酶II0.06mg，搅拌反应8小时，离心去沉淀，将上清液用板框过滤器过滤，收集得第一滤液200μl；
b)氧化：控制步骤a)所得第一滤液温度至30℃，用20％(w/v)氢氧化钙溶液调整第一滤液pH值至9，加入1μl过氧化氢，室温搅拌反应6小时，得纯化液；
c)超滤除杂：将步骤b中所获得的纯化液用4mol/L的盐酸调节为pH为6后进行切向流超滤，选择截留分子量为3,000Da的超滤膜进行循环超滤8小时，收集得第一截留液120μl；
d)醇沉除杂：向步骤c)所得的第一截留液中加入1.44mg氯化钠，搅拌溶解后再加入96μl 95％(v/v)乙醇，沉淀10小时，分离得中间沉淀物；中间沉淀物加160μl水溶解，并向溶液中加入192μl 95％(v/v)乙醇，沉淀8小时，分离得第一沉淀物，第一沉淀物真空干燥，重18g；
e)降解：向步骤d)中所得的第一沉淀物中加入144mg 2％(v/v)的冰醋酸溶解，溶解得第二料液，用4mol/L的盐酸调节第二料液pH值至3.0，调整温度至18℃时，加入0.38mg亚硝酸钙，搅拌反应25分钟，得到降解液；
f)还原：反应完成后，迅速用20％(w/v)氢氧化钙溶液调节降解液的pH值至9.0，加 入0.16mg硼氢化钠，搅拌反应10h，反应完成后得第三料液，用4mol/L的盐酸调节第三料液的pH值至3.0，搅拌反应15分钟后，得第四料液，用20％(w/v)氢氧化钠溶液调节第四料液的pH值至6，然后加入1.26mg氯化钠，搅拌溶解，然后用微孔过滤器过滤，得第二滤液，并向第二滤液中加入260μl 95％(v/v)乙醇，搅拌30min后，静置8小时；去上清，得第二沉淀物；
g)紫外照射：向步骤f)中的第二沉淀物加入140mg水搅拌使其溶解，在室温下用254nm的波长的紫外线照射20分钟，得第五料液；
h)氧化：用20％(w/v)氢氧化钙溶液调节第五料液pH值至9.0，加入1.1μl的过氧化氢，调节第五料液的温度至20℃，搅拌反应6小时；氧化完毕后，用4mol/L的盐酸调节料液pH值至7.2，向其中加入1.12mg氯化钠及360μl 95％(v/v)乙醇，搅拌20分钟以后，静止沉淀8小时，去上清，得第三沉淀物，向第三沉淀物中加入160mg水进行溶解；
i)钙置换：在搅拌状态下持续加入1.28mg氯化钙，25℃下搅拌反应10小时，得置换液；
j)纳滤：反应完成，滤板过滤，将置换液加入纳滤机开始循环纳滤，中间补加适量的水，直到纳滤截留液的体积浓度达到20％时，放出第二截留液，加入320μl 95％(v/v)乙醇，然后静置沉淀13小时制得钙盐沉淀物；
k)超滤精制：向步骤j)中所得的钙盐沉淀物加入156mg水进行溶解，完全溶解后先用0.22um的滤膜过滤，然后用2000Da的膜循环超滤控制分子量，收集第三截留液；将第三截留液经0.1μm的除菌膜过滤后，得第三滤液，再用1mol/L的盐酸溶液调节第三滤液pH值至6.5；
L)将第三滤液转移至真空冷冻干燥箱中进行冻干处理，得低分子肝素那曲肝素钙标准品库成品15.4mg。
制备实施例四：
本实施例提供了一种低分子肝素那曲肝素钙标准品库的制备方法，其包括以下具体步骤：
a)酶解：取粗品肝素钠20mg，并向其中加入220mg水溶解，溶解得第一料液，用20％(w/v)氢氧化钙溶液调节第一料液pH值至8.5，调节第一料液温度至40℃，分别加入木瓜蛋白酶0.12mg、核糖核酸酶II0.1mg，搅拌反应9小时，离心去沉淀，将上清液用板框过滤器过滤，收集得第一滤液220μl。；
b)氧化：控制步骤a)所得第一滤液温度至40℃，用20％(w/v)氢氧化钙溶液调整第一滤液pH值至11，加入0.7μl过氧化氢，室温搅拌反应7小时，得纯化液；
c)超滤除杂：将步骤b中所获得的纯化液用4mol/L的盐酸调节pH为8后进行切向流超滤，选择截留分子量为6,000Da的超滤膜进行循环超滤5小时，收集得第一截留液140μl；
d)醇沉除杂：向步骤c)所得的第一截留液中加入1.68mg氯化钠，搅拌溶解后再加入113μl 95％(v/v)乙醇，沉淀11小时，分离得中间沉淀物；中间沉淀物加150μl水溶解，并向溶液中加入180μl 95％(v/v)乙醇，沉淀8小时，分离得第一沉淀物，第一沉淀物真空干燥，重17.9mg；
e)降解：向步骤d)中所得的沉淀物中加入143.2mg 2％(v/v)的冰醋酸溶解，溶解得第二料液，用4mol/L的盐酸调节第二料液pH值至3.2，调整温度至22℃时，加入0.38mg亚硝酸钙，搅拌反应25分钟，得到降解液；
f)还原：反应完成后，迅速用20％(w/v)氢氧化钙溶液调节降解液的pH值至10.0，加入0.18mg硼氢化钠，搅拌反应15h，反应完成后得第三料液，用4mol/L的盐酸调节第三料液的pH值至4.0，搅拌反应30分钟后，得第四料液，用20％(w/v)氢氧化钠溶液调节第四料液的pH值至8，然后加入1.4mg氯化钠，搅拌溶解，然后用微孔过滤器过滤，得第二滤液，并向第二滤液中加入340μl 95％(v/v)乙醇，搅拌20min后，静置12小时；去上清，得第二沉淀物；
g)紫外照射：向步骤f)中的沉淀物去上清，加入152mg水搅拌使其溶解，在室温下用254nm的波长的紫外线照射30分钟，得第五料液；
h)氧化：用20％(w/v)氢氧化钙溶液调节第五料液pH值至10.0，加入1.2μl的过氧化氢，调节第五料液的温度至30℃，搅拌反应6小时；氧化完毕后，用4mol/L的盐酸调节料液pH值至7.0，向其中加入1.2mg氯化钠及400μl 95％(v/v)乙醇，搅拌20分钟以后，静止沉淀9小时，去上清，得第三沉淀物，向第三沉淀物中加入160mg水进行溶解；
i)钙置换：在搅拌状态下持续加入1.28mg氯化钙，25℃下搅拌反应11小时，得置换液；
j)纳滤：反应完成，滤板过滤，将置换液加入纳滤机开始循环纳滤，中间补加适量的水，直到纳滤截留液的体积浓度达到23％时，放出第二截留液，加入400μl 95％(v/v)乙醇，然后静置沉淀12小时制得钙盐沉淀物；
k)超滤精制：向步骤j)中所得的钙盐沉淀物加入1600mg水进行溶解，完全溶解后先用0.22um的滤膜过滤，然后用3000Da的膜循环超滤控制分子量，收集第三截留液；将第三截留液经0.1μm的除菌膜过滤后，得第三滤液，再用1mol/L的盐酸溶液调节第三滤液pH值至7.2；
L)将第三滤液转移至真空冷冻干燥箱中进行冻干处理，得低分子肝素那曲肝素钙标准品库成品15.3mg。
试验例：
分别以制备实施例一～四所得低分子肝素那曲肝素钙标准品库成品为样品一、样品二进行实验分析，与国家和进口标准有关数据的对比情况如表一所示。
表一：
Figure PCTCN2017090904-appb-000001
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
工业实用性
本发明的低分子肝素那曲肝素钙标准品库及其制备方法例如包括以下突出的有益效果：
(一)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，对粗品肝素钠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；
(二)进行亚硝酸盐降解前，将经过处理的肝素钠加入具有缓冲作用的冰醋酸，克服了传统工艺与亚硝酸盐反应时的pH变化剧烈而引起的产品分子量剧烈变化，生产产品结构不 合格，产品稳定性差等缺陷，增加产品的稳定性；
(三)在一定温度和酸性条件下，与亚硝酸钙发生降解反应。采用亚硝酸钙降解，会有部分钙离子置换出产品中的钠离子，这样可以适当减少后期钙置换时氯化钙的投入，及其钙置换的反应时间，降低了成本投入和缩短了制备周期；
(四)将先进的纳滤技术应用于产品中游离钙离子的去除，改变了落后的溶媒分级沉淀去除法，产品收率大幅提高；
(五)由于本实施例中的目的在于生产标准品，主要用于研究而非生产，因此该工艺的成本投入以及收率等因素不作为本实施例的主要考量对象，最终成品的品质和质量作为最主要的考量对象，本实施例中，通过本发明方法制备得到的低分子肝素那曲肝素钙标准品库成品的平均分子量为3800～5000道尔顿，分子量分布为：分子量小于2000的比率为≤15.0％；分子量于2000～8000的比率为75.0％～95.0％；分子量在2000～4000的比率为35.0％～55.0％，抗FXa因子活性为100IU/mg～130IU/mg，抗FXa/FIIa比值为2.7～4.0，pH值为6.5～7.2；5％的产品水溶液澄清透明；乙醇含量≤0.8％；钙含量(干品)9.7％～11.3％，产品质量超越国内标准和欧洲药典标准，生物利用度高、体内半衰期长、出血倾向小；品质高、质量高；
(六)操作简单，制得的成品的品质高、质量高。

Claims (16)

  1. 一种低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，包括以下步骤：
    a)酶解：向粗品肝素钠中加入10～12倍质量的水溶解，溶解得第一料液，调节所述第一料液的pH值至8.0～8.5，温度至35～40℃，加入木瓜蛋白酶及核糖核酸酶II，酶解反应8～10小时，离心去沉淀，上清液过滤，收集第一滤液；
    b)氧化脱色：控制所述第一滤液温度至30～40℃，调整所述第一滤液pH值至9～11，加入0.3％～0.5％所述第一滤液体积的过氧化氢，搅拌反应6～8小时，得纯化液；
    c)超滤除杂：调节所述纯化液pH值至6～8，采用截留分子量为3,000～8,000Da的超滤膜对所述纯化液进行切向流循环超滤，循环超滤5～8小时，收集第一截留液；
    d)醇沉除杂：所述第一截留液经醇沉除杂，得到第一沉淀物，对所述第一沉淀物干燥称重；
    e)降解：向所述第一沉淀物中加入7～8倍质量的冰醋酸溶解，溶解得第二料液，调节所述第二料液pH值至3.0～3.2，调整所述第二料液温度至18～22℃，加入亚硝酸盐，搅拌反应20～30分钟，得到降解液；所述冰醋酸的体积浓度为1.8～2.0％；所述亚硝酸盐的用量为所述第一沉淀物重量的1.5％～2.1％；
    f)还原：降解反应完成后，调节所述降解液的pH值至9.0～10.0，加入所述第一沉淀物重量0.9％～1.1％的硼氢化钠，搅拌反应10～15h，反应完成后得第三料液，调节所述第三料液的pH值至3.0～4.0，搅拌反应15～30分钟后，得第四料液，再调节所述第四料液的pH值至6～8，然后加入氯化钠，搅拌溶解，微孔过滤器过滤，得第二滤液，并向所述第二滤液中加入2～3倍体积的95％乙醇，搅拌20～30min后，静置8～12小时；去上清，得第二沉淀物；氯化钠与所述第四料液的质量体积比为0.9％～1.1％；
    g)紫外照射：向所述第二沉淀物中加入8～10倍质量的水搅拌使其溶解，在室温下用254nm的波长的紫外线照射20～30分钟，得第五料液；
    h)氧化：调节所述第五料液pH值至9～10，加入0.8％～1.0％料液体积的过氧化氢，调节所述第五料液的温度至20～30℃，搅拌反应6～8小时；氧化完毕，醇沉，静止沉淀8～10小时后，去上清，得第三沉淀物，向所述第三沉淀物中加入9～10倍质量的水溶解；
    i)钙置换：在搅拌状态下持续加入氯化钙，20～25℃下搅拌反应10～12小时，得置换液；所述氯化钙的用量为所述第三沉淀物重量的8～12％；
    j)纳滤：反应完成，所述置换液经过滤后加入纳滤机开始循环纳滤，纳滤截留液的体积浓度达到20％～25％时，放出第二截留液，加入所述第二截留液体积2～3倍量的 95％乙醇，静置沉淀12～15小时制得钙盐沉淀物；
    k)超滤精制：向所述钙盐沉淀物加入10～12倍质量的水进行溶解，完全溶解后先用0.22um的滤膜过滤，然后用1000～3000Da的膜循环超滤控制分子量，收集第三截留液；所述第三截留液经0.1μm的除菌膜过滤，收集第三滤液，并调节所述第三滤液pH值至6.5～7.2；
    l)将所述第三滤液进行冻干处理，得低分子肝素那曲肝素钙标准品库成品。
  2. 根据权利要求1所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，所述木瓜蛋白酶的用量为所述粗品肝素钠重量的0.5％～0.7％，所述核糖核酸酶II的用量为所述粗品肝素钠重量的0.3％～0.5％。
  3. 根据权利要求1所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，步骤d中醇沉除杂时，向所述第一截留液中加入氯化钠，搅拌溶解后再加入0.8～1.0倍体积的95％乙醇，沉淀10～12小时，分离得中间沉淀物；再将所述中间沉淀物加水溶解，向溶液中加入1～1.2倍体积的95％乙醇，沉淀6～8小时，分离得所述第一沉淀物，所述第一沉淀物真空干燥并称重；氯化钠与第一截留液的质量体积比为1.0％～1.2％。
  4. 根据权利要求1所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，步骤e中所述亚硝酸盐为亚硝酸钙。
  5. 根据权利要求1所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，步骤h中醇沉的具体过程为：调节所述置换液pH值至6～8，向所述置换液中加入氯化钠及2～3倍体积的95％乙醇；氯化钠与所述置换液的质量体积比为0.8％～1.0％。
  6. 一种低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，包括以下步骤：
    向粗品肝素钠的水溶液中加入木瓜蛋白酶及核糖核酸酶II，酶解后离心去沉淀，收集第一滤液；
    所述第一滤液经除杂后，得第一沉淀物；
    向所述第一沉淀物中加入冰醋酸溶解，调节pH至3.0～3.2，调节温度至18～22℃，接着加入亚硝酸盐进行降解，搅拌反应20～30分钟，得降解液。
  7. 根据权利要求6所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，所述冰醋酸的浓度为1.8～2.0％；所述冰醋酸的加入量为所述第一沉淀物的质量的7～8倍；所述亚硝酸盐的用量为所述第一沉淀物重量的1.5％～2.1％。
  8. 根据权利要求6所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，在对所述第一滤液进行除杂之前，还包括对所述第一滤液进行氧化脱色：控制所 述第一滤液温度至30～40℃，调整所述第一滤液pH值至9～11，加入0.3％～0.5％所述第一滤液体积的过氧化氢，搅拌反应6～8小时，得纯化液。
  9. 根据权利要求8所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，除杂所述第一滤液包括：调节所述纯化液pH值至6～8，采用截留分子量为3,000～8,000Da的超滤膜对所述纯化液进行切向流循环超滤除杂，循环超滤5～8小时，收集第一截留液；所述第一截留液经醇沉除杂，得到所述第一沉淀物，对所述第一沉淀物干燥称重。
  10. 根据权利要求6所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，在制得所述降解液之后，还包括还原所述降解液步骤：调节所述降解液的pH值至9.0～10.0，加入硼氢化钠，搅拌反应10～16h，调节pH值至6～8，然后加入氯化钠，搅拌溶解，过滤后向滤液中加入乙醇，搅拌20～30min后，静置8～12小时；去上清，得第二沉淀物。
  11. 根据权利要求10所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，在还原所述降解液步骤之后，还包括：将所述第二沉淀物经紫外照射、氧化和钙置换后，得置换液，将所述置换液经过滤后进行循环纳滤，纳滤截留液的体积浓度达到20％～25％时，放出第二截留液，加入第二截留液体积2～3倍量的95％乙醇，静置沉淀12～15小时制得钙盐沉淀物。
  12. 根据权利要求11所述的低分子肝素那曲肝素钙标准品库的制备方法，其特征在于，在制得所述钙盐沉淀物之后，还包括：向所述钙盐沉淀物加入10～12倍质量的水进行溶解，完全溶解后先用0.2～0.3um的滤膜过滤，然后用1000～3000Da的膜循环超滤控制分子量，收集第三截留液；所述第三截留液经0.1～0.2μm的除菌膜过滤，收集第三滤液，并调节所述第三滤液pH值至6.5～7.2；将所述第三滤液进行冻干处理。
  13. 一种低分子肝素那曲肝素钙标准品库，其特征在于，其是由权利要求1～12任一项所述的低分子肝素那曲肝素钙标准品库的制备方法制得。
  14. 根据权利要求13所述的低分子肝素那曲肝素钙标准品库，其特征在于，所述低分子肝素那曲肝素钙标准品库的平均分子量为3800～5000道尔顿。
  15. 根据权利要求13所述的低分子肝素那曲肝素钙标准品库，其特征在于，所述低分子肝素那曲肝素钙标准品库的分子量分布为：分子量小于2000的比率为≤15.0％；分子量于2000～8000的比率为75.0％～95.0％；分子量在2000～4000的比率为35.0％～55.0％。
  16. 根据权利要求13所述的低分子肝素那曲肝素钙标准品库，其特征在于，所述低分子肝素那曲肝素钙标准品库的抗FXa因子活性为100IU/mg～130IU/mg、pH值为 6.5～7.2、抗FXa/FIIa比值为2.7～4.0。
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