CN104098716B - Production method of dalteparin sodium fine product - Google Patents
Production method of dalteparin sodium fine product Download PDFInfo
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Abstract
The invention discloses a production method of dalteparin sodium. According to the production method, a heparin sodium coarse product is taken as a raw material, a low-molecular-weight heparin sodium coarse product is obtained through degradation, reduction and ethanol precipitation; compared with a method taking a heparin sodium fine product as the raw material, the method is wider in application range and can better control the quality of a dalteparin sodium fine product from the source. Innovatively, molecular weight and distribution of the low-molecular-weight heparin sodium coarse product before chromatography are tested through HPLC (high performance liquid chromatography), corresponding required-to-be-removed areas of macromolecules and micromolecules and corresponding volumes are calculated according to the formula, filtrate in the chromatography process is collected, a low-molecular-weight heparin sodium fine product liquid which meets the requirements is accurately and quantitatively obtained, and use of an expensive molecular weight interception filtering membrane is avoided; a one-pot process is achieved through oxidation, sterilization filtration and sediment dehydration, the operation is simplified, and time and labor cost are saved; and a new method for preparing a raw dalteparin sodium material is developed on the basis of the prior art, and industrial production of dalteparin sodium is realized.
Description
Technical field
The present invention relates to biomedicine field, particularly a kind of preparation of fine work dalteparin sodium and purifying process.
Background technology
Heparin sodium (Heparin Sodium) is mucopolysaccharide sulfuric acid ester anticoagulant.Heparin sodium is the sodium salt of the CSSO3 extracted in the intestinal mucosa by pig or ox, belongs to mucopolysaccharide material.All having blood coagulation resisting function in vivo and in vitro, is the anticoagulation medicine of current main flow.But the bioavailability of unfractionated heparin sodium is low, side effect is large, and it is found that the sodium salt of the CSSO3 fragment that heparin sodium cracking obtains afterwards, molecular-weight average is 4000 ~ 6000 dalton, is called low molecular sodium heparin.Low molecular heparin sodium injection is the same with heparin sodium injection belongs to antithrombin Ⅲ (AT III) dependency thrombin inhibitors.But compared with heparin sodium injection, have that the transformation period is longer, antithrombotic effect good, bleeding tendency is more weak, convenient drug administration, but price comparison is expensive.And DALT is wherein a kind of.Dalteparin sodium injection liquid is mainly used in the treatment of acute deep venous thrombosis, prevent from, in extracorporeal circulation system, blood coagulation occurs during acute renal failure or chronic renal insufficiency person carry out hemodialysis and blood filtration, instability mode coronary heart disease, as unstable angina pectoris and non-Q-wave mode myocardial infarction, prevent the thrombosis relevant with operation.It is the anti-thrombotic drugs of current main flow.
Existing 3 sections patent reports preparation process of dalteparin sodium at present, wherein have 2 sections to authorize.Patent CN101942038A passes through heparin sodium material dissolution in purified water with containing in the organic solvent of methyl, under certain temperature and pH value condition, add Sodium Nitrite to degrade, after reduce with sodium hydroxide under certain pH value condition again, then use alcohol settling, membrane filtration, ultraviolet disinfection, hydrogen peroxide oxidation membrane filtration, then use alcohol settling, namely throw out lyophilize or dewatering and vacuum drying are obtained dalteparin sodium finished product.Its Patent CN102558393A adopts the preparation technology of the most traditional dalteparin sodium, namely first heparin sodium is dissolved in purified water and obtains the heparin sodium aqueous solution, the pH value of hydrochloric acid regulating solution, to degrade heparin sodium with Sodium Nitrite afterwards, use sodium hydroxide regulation system pH value again, sodium borohydride reduction, rear alcohol settling obtains precipitate reduction thing, again with hydrogen peroxide oxidation, crude product dalteparin sodium is obtained to it, then after carrying out ultrafiltration with ultra-filtration membrane, freeze-drying obtains fine work dalteparin sodium finished product.
Above-mentioned 2 sections of patent basic ideas of having authorized are consistent, but all have certain disadvantages, and patent CN101942038A is raw material with refined heparin sodium in preparation process, is difficult to the quality from Sources controlling heparin sodium, and adds the cost preparing dalteparin sodium.In preparation process, add the organic solvent containing methyl, cause and need to control organic solvent residual in final dalteparin sodium raw material standard, increase testing amount and the quality reducing product, with lack degradation condition system, accurate control, thus cause the Low molecular heparin purity for preparing inadequate, molecular weight distribution is not concentrated, the shortcoming that impurities left is too much, these all current techniques cann't be solved.There is certain shortcoming in the preparation of supply injection medicine.Patent CN102558393A has carried out certain simplification on the basis of that patent CN101942038A above-mentioned, first the use of the organic solvent containing methyl is eliminated, decrease the consumption of purified water, must filter after decreasing filtration and the degerming operation steps of ultraviolet and hydrogen peroxide oxidation, directly use molecular cut-off membrane filtration, obtain dalteparin sodium fine work.This patent is raw material equally with refined heparin sodium, and on producing, the acquisition of raw material is subject to certain restrictions like this, and adds cost, is difficult to the production technique from the whole dalteparin sodium of Sources controlling and quality product simultaneously.Employ expensive molecular cut-off filtering membrane, molecular cut-off filtering membrane uses frequent, adds technology production cost simultaneously.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the invention provides a kind of production method of dalteparin sodium fine work, the method uses heparin sodium crude can obtain quality good dalteparin sodium fine work for raw material and obtain higher yield, achieves dalteparin sodium low cost, high efficiency suitability for industrialized production.
A kind of technique preparing dalteparin sodium raw material provided by the invention, it comprises the steps:
(1) preparation of low molecular sodium heparin crude product
Taking heparin sodium crude product adds purified water and dissolves.Regulator solution pH to 2.6-2.8, adds Sodium Nitrite, maintains the temperature at 15-25 DEG C and stirs 20min, then regulator solution pH to 9.5-10.0, and add sodium borohydride stirring >=15h, hydrochloric acid conditioning solution pH to 3.4-3.6 stirs 20min.Sodium hydrate regulator solution pH7.0-7.5,254nm UV illumination penetrates degradation solution 20-25min, adds alcohol settling, and leave standstill more than 8h, put into vacuum drying oven after the solid after precipitation dewaters again, 60-65 DEG C of dry 40-48h, obtains low molecular sodium heparin crude product.
(2) low molecular sodium heparin fine work preparation (gel filtration chromatography)
Add in chromatography column after above-mentioned low molecular sodium heparin crude product is dissolved with 2% sodium chloride solution, at the uniform velocity wash with 2% sodium chloride solution and open up, receive solution, measure the ultraviolet OD200 absorbance value of each glass, draw collection of illustrative plates according to numerical value.By the molecular chain conformation data of HPLC test, determine that the large and small molecular percentage that need be removed by gel chromatography is than number, go out the large and small molecular area of corresponding need removing and corresponding volume by formulae discovery.Collect the solvent portions of needs and regulate pH to 7.0-7.5, alcohol settling leaves standstill >=8h, wet solid 60-65 DEG C of vacuum-drying 18-24h after dehydration.
(3) mixed dissolution
The low molecular sodium heparin fine work gone out by batch-type merges, and test its molecular chain conformation, if small molecules part has deviation, adjustable pH value of solution 7.0-7.5, adds alcohol settling and regulate small molecule segment, makes it reach and meets the requirements.Low molecular sodium heparin fine work qualified for molecular chain conformation is added in 13# oxidation tank, adds purified water and sodium-chlor, stirring and dissolving.
(4) hydrogen peroxide oxidation
Regulate pH value of solution 10.5-11.0 in above-mentioned 13# oxidation tank, add 30% hydrogen peroxide oxidation >=4-5h.Regulate pH7.9-8.1 after end, add after sodium sulphite anhydrous 99.3 stirs 30min and filter, obtain dalteparin sodium crude product solution.
(5) Sterile Filtration
Regulate dalteparin sodium crude product solution pH5.6-5.8, by 0.45 μm and 0.2 μm of filter membrane series winding, 0.45 μm of port is connected with the outlet of 13# oxidation tank, open retort outlet valve, make pot liquid successively by 0.45 μm and 0.2 μm of filtering membrane, the dalteparin sodium solution collection after Sterile Filtration is in clean setting tank.
(6) preparation of dalteparin sodium fine work
Dalteparin sodium solution after filtration adds alcohol settling and leaves standstill >=more than 8h.Rear filtration also obtains fine work dalteparin sodium with ethanol dehydration, centrifuge dewatering vacuum-drying.
In step (1), heparin sodium crude and purified water feed ratio (W/V) are 1.0:8.5-10, are preferably 1.0:9.2-9.5.The feed ratio (W/W) of heparin sodium crude and Sodium Nitrite is 100:1.5-2.5, is preferably 100:1.5-2.0.The feed ratio (W/W) of Sodium Nitrite and sodium borohydride is 1.0:0.3-0.5, is preferably 1.0:0.35-0.42.During Sodium Nitrite reaction, system temperature maintains 15-30 DEG C, and 4mol/L hydrochloric acid regulates pH2.6-2.8, by determining that with starch-kalium iodide test paper test soln reaction terminates, 5mol/L sodium hydrate regulator solution pH to 9.0-10.5.Solution after 254nm ultraviolet lamp is placed in degraded carries out sterilizing, adds liquor capacity and ethanol contend (V/V) is 1.0:0.65-0.75, and preferred 0.65-0.70 precipitates.The low molecular sodium heparin crude product be settled out needs to use ethanol dehydration again three times in production, the ethanol of first time is 3.0-5.0:1.0 with the ratio (V/W) of the heparin sodium crude fed intake, be preferably 3.5-4.0:1.0, the ethanol of second time and third time is 50% of front ampoule, each dehydration interval more than 2h, and molecular chain conformation test is carried out to the Low molecular heparin sodium sample obtained.
Need in step (2) with 2% sodium chloride solution balance chromatography column 1-2h, flow velocity is about column volume/2 hour.The low molecular sodium heparin crude product that sodium chloride solution dissolving step (1) with 2% obtains, makes final solution volume and low molecular sodium heparin crude product weight (V/W) be 7.0-8.5:1.0, is preferably 7.5-8.0:1.0.Above-mentioned solution adds in chromatography column by constant flow pump, ensures the smooth of gel interface in application of sample process.After adding sample, at the uniform velocity wash with 2% sodium chloride solution and open up gel column, go out by formulae discovery below the large and small molecular area and corresponding volume that corresponding need remove by the HPLC molecular chain conformation test result of crude product above.
A. set the per-cent of the macromolecule fraction area of removing as X%
the macromole content %-X% of low molecule raw material=(15 ~ 25) %
100％-X％
X%=
macromole content %-(15 ~ 25) % of low molecule raw material
100％-(15～25)％
＝____________＝____________＝__________％
Formula one
B. set the per-cent of the small molecules area of removing as Y%
the small molecules content %-Y% of low molecule raw material=-≤13%
100％-Y％
Y%=
small molecules content %+≤13% of low molecule raw material
100％+≤13％
＝__________＝____________＝__________％
Formula two
The centre of collecting is washed the ethanol opening up its 2 times of volumes of liquid and is precipitated, throw out dewaters three times with ethanol again, the ethanol of first time is 3.0-5.0:1.0 with the ratio (V/W) of the low molecular sodium heparin crude product fed intake, be preferably 3.5-4.0:1.0, the ethanol of second time and third time is 50% of front ampoule, each dehydration interval more than 2h.
The low molecular sodium heparin mainly gone out chromatography in step (3) carries out the test of molecular weight and molecualr weight distribution, if result display small molecules part (<3000) tested out does not meet the requirement of≤13.0%, can dissolve chromatography low molecular sodium heparin fine work purified water out, and regulate pH7.0-7.5, alcohol settling, makes it reach and meets the requirements.Be 1.0:8.5-10 by low molecular sodium heparin fine work qualified for molecular chain conformation and purified water (W/V), be preferably 1.0:9.2-9.5, sodium-chlor and purified water (W/V) are 0.8-1.5:100, are preferably 0.9-1.2:100, are contained in 13# oxidation tank.
By the solution sodium hydrate regulator solution pH10.5-11.0 in above-mentioned steps (3) in step (4), 30% hydrogen peroxide added and solution (V/V) are 0.25-0.45:100, be preferably 0.25-0.3:100, oxidization time is 4-5h.Sodium sulphite anhydrous 99.3 and hydrogen peroxide (W/V) are 0.20-0.30:1.0, are preferably 0.20-0.25:1.0.
Need to do 0.2 μm and 0.45 μm of integrity of filtration membranes test in step (5).Guarantee that the filter membrane used in producing can meet Sterile Filtration requirement.
Dalteparin sodium solution after filtering in step (6) alcohol settling of 2.0 times of volumes.The dalteparin sodium fine work ethanol dehydration be settled out three times, the ethanol of first time is 3.0-5.0:1.0 with the ratio (V/W) of the low molecular sodium heparin fine work fed intake, be preferably 3.5-4.0:1.0, the ethanol of second time and third time is 50% of front ampoule, each dehydration interval more than 2h.
The present invention be directed to existing dalteparin sodium production technique to be optimized and to improve, use heparin sodium crude is raw material, now obtain low molecular sodium heparin crude product by degraded, reduction, alcohol settling, refined heparin sodium is used to be raw material relative to needs, the method scope of application is wider, more can from the quality of Sources controlling dalteparin sodium fine work.The collection going out filtrate in chromatography process by testing its molecular chain conformation and formulae discovery to the HPLC of the low molecular sodium heparin crude product before chromatography of novelty, determine to wash and which to be opened up in liquid need to retain, which needs to give up, and very effectively also accurately quantitatively to satisfactory low molecular sodium heparin fine work liquid, avoid the use of expensive molecular cut-off filtering membrane, this is unexistent in existing technology.Low molecular sodium heparin fine work obtains dalteparin sodium crude product by oxidizing reaction, and reaction solution obtains dalteparin sodium fine work by filtration, precipitation, drying.Dewater achieve the technique of " treating different things alike " from oxidation, Sterile Filtration, precipitation, operationally achieve simplification, save time and human cost, this technique have developed a new method preparing dalteparin sodium fine work in existing technical foundation, and achieves the suitability for industrialized production of dalteparin sodium in our company.
Embodiment
In order to make those skilled in the art person understand technical scheme of the present invention better, below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
Add 90L water and heparin sodium crude 10kg in retort, stirring makes it dissolve completely, be 2.7 with solution is pH value in 4mol/L hydrochloric acid regulating tank, take 0.18kg Sodium Nitrite and add in beaker, in beaker, add 1620mL purified water, after stirring and dissolving, add in retort, maintaining solution ph in tank is 2.6-2.8, with starch-kalium iodide test paper test soln after stirring reaction 20min, observe test paper color, till the not aobvious blueness of test paper.Be 9.6 by 5mol/L sodium hydroxide solution regulation system pH value, add 0.072kg sodium borohydride in retort, stirring reaction 16h, then be 3.5 by 4mol/L hydrochloric acid conditioning solution pH value, stir 20min.Use 5mol/L sodium hydroxide solution regulation system pH value to 7.5 again, the ultraviolet lamp of wavelength 254nm is switched on power, put into retort internal radiation 20min, liquor capacity is 132L, so 88.4L ethanol is slowly poured in retort, limit edged stirs, stir, precipitation leaves standstill 10h, remove supernatant liquor, 40L ethanol dehydration is added again in throw out, stir 2h and remove supernatant liquor, throw out adds 20L ethanol dehydration again, supernatant liquor is removed after 2h, throw out adds 20L ethanol dehydration again, supernatant liquor is removed after 2h, wet solid after dehydration is loaded in Stainless Steel Disc, put into vacuum drying case 60 ~ 65 DEG C of dry 48h, degradation product is weighed and obtains 7.15kg low molecular sodium heparin crude product, yield 71.50%.HPLC molecular chain conformation: molecular weight is 6550, >8000 part is 26.51% (answering 10.0-30.0%), <3000 part 15.5% (Ying≤30.0%), N-NO remains <0.25ppm (Ying≤0.25ppm).
Weighing 3kg sodium-chlor dissolves in purified water, and stirring and dissolving makes final volume be 150L, obtains the sodium chloride solution of 2%, weigh the low molecular sodium heparin crude product 0.4kg after degraded, add the sodium chloride solution of 2%, stirring and dissolving makes final volume be 3200mL, and regulator solution pH is 7.0, open constant flow pump, with the sodium chloride solution balance chromatography column 1-2h of 2%, equilibrium velocity is 145mL/min, balances complete, when sodium chloride solution trickling is to 1-2cm place in gel interface, close constant flow pump and outlet valve.Slowly added in chromatography column by solution constant flow pump after dissolving, first added-time flow velocity can not be too fast, prevents from destroying the smooth of gel interface.When solution adds to about 5cm on gel interface, open outlet valve, accelerate the flow velocity of application of sample simultaneously, until sample adds.When the trickling of low molecular sodium heparin crude product solution is in gel interface during 1-2cm place, start to connect sample.Meanwhile, carefully 2% sodium chloride solution is slowly pumped into constant flow pump.Receive 60 glasss of solution altogether, cumulative volume 30L, according to HPLC molecular chain conformation, the total area 65.29, macromolecule fraction is about 6.79, small molecules area about 13.71.The macromolecule fraction area being calculated removing by formula one is 10.4%, volume is 6.5L, the small molecules area that formula two calculates removing is 21%, volume is 10.75L, such intermediate collection area about 44.79, account for the total area 68.6%, volume 12.75L, therefore need to remove first 13 glasss and latter 21.5 glasss, wash to open up in liquid to the middle 12.75L collected and add 25.5L ethanol, limit edged stirs, stir, precipitation leaves standstill 14h, remove supernatant liquor, 28.6L ethanol dehydration is added again in throw out, stir 2h and remove supernatant liquor, throw out adds 14.3L ethanol dehydration again, supernatant liquor is removed after 2h, throw out adds 7.2L ethanol dehydration again, supernatant liquor is removed after 2h, wet solid after dehydration is loaded in Stainless Steel Disc, put into vacuum drying case 60-65 DEG C of dry 48h, weigh and obtain low molecular sodium heparin fine work 0.31kg, yield 77.50%.
With above-mentioned chromatography by the low molecular sodium heparin purifying crude of other 6.75kg out, obtain low molecular sodium heparin fine work 5.41kg altogether, yield 75.66%.Low molecular sodium heparin fine work test molecule amount after being combined and distribution, meet the requirements.By low molecular sodium heparin fine work 5.41kg, 0.49kg sodium-chlor and 48.69L purified water add in 13# oxidation tank, stirring and dissolving.
Liquor capacity is 51.64L, with 5mol/L sodium hydrate regulator solution pH10.8.Add 155mL30% hydrogen peroxide, oxidization time 4-5h, test per hour pH value, maintenance system pH value is within the scope of 10.5-11.0.With 4mol/L hydrochloric acid conditioning solution pH8.0, add the reduction of 38.75g sodium sulphite anhydrous 99.3, stir 30min.
By the solution ph to 5.8 that 4mol/L hydrochloric acid regulates above-mentioned reaction complete, rinse 0.45um filter membrane to neutral by purified water, then itself and 13# oxidation tank are exported correct installation.Integrity test is carried out to 0.2 μm of filter membrane, detect qualified after be arranged on fixed position between 0.45um filter membrane and clean area setting tank, then start to filter, filtrate is placed in clean setting tank, the sodium-chlor having filtered rear 10.4L1% rinses oxidation tank and also puts into setting tank, and obtaining dalteparin sodium crude product solution cumulative volume is 63L.
Open whipping appts in setting tank, 126L ethanol is slowly added in setting tank, ethanol adds rear continuation and stirs 3-5min, then 14h is staticly settled, incline supernatant liquor, add 21.64L ethanol and stir dehydration to fine particulate, leave standstill 2h, vacuum pumps supernatant liquor, add 10.82L ethanol and stir dehydration to fine particulate, leave standstill 2h vacuum and pump supernatant liquor, add 5.41L ethanol and stir dehydration to fine particulate, leave standstill 2h vacuum and pump supernatant liquor, to be dispensed in Stainless Steel Disc after dalteparin sodium throw out centrifuge dewatering 15min, a vacuum-drying 45-75 DEG C 40-44h obtains 4.12kg dalteparin sodium fine work, yield 76.16%.
Embodiment 2
Add 85L water and heparin sodium crude 10kg in retort, stirring makes it dissolve completely, be 2.7 with solution is pH value in 4mol/L hydrochloric acid regulating tank, take 0.15kg Sodium Nitrite and add in beaker, in beaker, add 1650mL purified water, after stirring and dissolving, add in retort, maintaining solution ph in tank is 2.6-2.8, with starch-kalium iodide test paper test soln after stirring reaction 25min, observe test paper color, till the not aobvious blueness of test paper.Be 9.8 by 5mol/L sodium hydroxide solution regulation system pH value, add 0.045kg sodium borohydride in retort, stirring reaction 18h, then be 3.5 by 4mol/L hydrochloric acid conditioning solution pH value, stir 23min.Use 5mol/L sodium hydroxide solution regulation system pH value to 7.3 again, the ultraviolet lamp of wavelength 254nm is switched on power, put into retort internal radiation 20min, liquor capacity is 126L, 82.55L ethanol is slowly poured in retort, limit edged stirs, stir, precipitation 10h, remove supernatant liquor, 30L ethanol dehydration is added again in throw out, stir 2h and remove supernatant liquor, throw out adds 15L ethanol dehydration again, supernatant liquor is removed after 2h, throw out adds 7.5L ethanol dehydration again, supernatant liquor is removed after 2h, wet solid after dehydration is loaded in Stainless Steel Disc, put into vacuum drying case 60-65 DEG C of dry 44h, degradation product is weighed and obtains 7.18kg low molecular sodium heparin crude product, yield 71.80%.HPLC molecular chain conformation: molecular weight is 6450, >8000 part is 26.50% (answering 10.0-30.0%), <3000 part 15.5% (Ying≤30.0%), N-NO remains <0.25ppm (Ying≤0.25ppm).
Weighing 3kg sodium-chlor dissolves in purified water, and stirring and dissolving makes final volume be 150L, obtains the sodium chloride solution of 2%, weigh the low molecular sodium heparin crude product 0.4kg after degraded, add the sodium chloride solution of 2%, stirring and dissolving makes final volume be 3200mL, and regulator solution pH is 7.0, open constant flow pump, with the sodium chloride solution balance chromatography column 1-2h of 2%, equilibrium velocity is 140mL/min, balances complete, when sodium chloride solution trickling is to 1-2cm place in gel interface, close constant flow pump and outlet valve.Slowly added in chromatography column by solution constant flow pump after dissolving, first added-time flow velocity can not be too fast, prevents from destroying the smooth of gel interface.When solution adds to about 5cm on gel interface, open outlet valve, accelerate the flow velocity of application of sample simultaneously, until sample adds.When the trickling of low molecular sodium heparin crude product solution is in gel interface during 1-2cm place, start to connect sample.Carefully slowly pump into 2% sodium chloride solution with constant flow pump simultaneously.Receive 60 glasss of solution altogether, cumulative volume 30L, according to HPLC molecular chain conformation, the total area 67.74, macromolecule fraction is about 7.04, small molecules area about 14.23.The macromolecule fraction area being calculated removing by formula one is 10.4%, volume is 7.75L, the small molecules area that formula two calculates removing is 21%, volume is 9.5L, such intermediate collection area about 46.47, account for the total area 68.6%, volume 12.75L, therefore need to remove first 15.5 glasss and latter 19 glasss.Wash to open up in liquid to the middle 12.75L collected and add 25.5L ethanol, limit edged stirs, stir the standing 14h of precipitation and remove supernatant liquor, 28.6L ethanol dehydration is added again in throw out, stir 2h and remove supernatant liquor, throw out adds 14.3L ethanol dehydration again, supernatant liquor is removed after 2h, throw out adds 7.2L ethanol dehydration again, remove supernatant liquor after 2h, the wet solid after dehydration is loaded in Stainless Steel Disc, puts into vacuum drying case 60-65 DEG C of dry 48h, weigh and obtain low molecular sodium heparin fine work 0.29kg, yield 72.50%.
With above-mentioned chromatography by the low molecular sodium heparin purifying crude of other 6.78kg out, obtain low molecular sodium heparin fine work 5.45kg altogether, yield 75.91%.Low molecular sodium heparin fine work test molecule amount after being combined and distribution, meet the requirements.By low molecular sodium heparin fine work 5.45kg, 0.37kg sodium-chlor and 46.33L purified water add in 13# oxidation tank, stirring and dissolving.
Liquor capacity is 49.24L, with 5mol/L sodium hydrate regulator solution pH10.5.Add 123mL30% hydrogen peroxide, oxidization time 4.4h, test per hour pH value, maintenance system pH value 10.5-11.0.With 4mol/L hydrochloric acid conditioning solution pH7.9, add the reduction of 24.60g sodium sulphite anhydrous 99.3, stir 30min.
By the solution ph to 5.6 that 4mol/L hydrochloric acid regulates above-mentioned reaction complete, rinse 0.45um filter membrane to neutral by purified water, then itself and 13# oxidation tank are exported correct installation.Integrity test is carried out to 0.2 μm of filter membrane, detect qualified after be arranged on fixed position between 0.45um filter membrane and clean area setting tank, then start to filter, filtrate is placed in clean setting tank, the sodium chloride solution having filtered rear 9.85L1% rinses oxidation tank, washing fluid also puts into setting tank, and obtaining dalteparin sodium crude product solution cumulative volume is 60.2L.
Open whipping appts in setting tank, 120.4L ethanol is slowly added in setting tank, ethanol adds rear continuation and stirs 3-5min, then 10h is staticly settled, incline supernatant liquor, add 16.35L ethanol and stir dehydration to fine particulate, leave standstill 2h, vacuum pumps supernatant liquor, add 8.18L ethanol and stir dehydration to fine particulate, leave standstill 2h, vacuum pumps supernatant liquor, add 4.09L ethanol and stir dehydration to fine particulate, leave standstill 2h vacuum and pump supernatant liquor, to be dispensed in Stainless Steel Disc after dalteparin sodium throw out centrifuge dewatering 15min, a vacuum-drying 45-75 DEG C 40-44h obtains 4.14kg dalteparin sodium fine work, yield 75.96%.
Embodiment 3
Add 100L water and heparin sodium crude 10kg in retort, stirring makes it dissolve completely, be 2.7 by solution ph in 4mol/L hydrochloric acid regulating tank, take 0.25kg Sodium Nitrite and add in beaker, in beaker, add 1550mL purified water, after stirring and dissolving, add in retort, maintaining solution ph in tank is 2.6-2.8, with starch-kalium iodide test paper test soln after stirring reaction 25min, observe test paper color, till the not aobvious blueness of test paper.Be 10.0 by 5mol/L sodium hydroxide solution regulation system pH value, add 0.125kg sodium borohydride in retort, stirring reaction 16h, then be 3.6 by 4mol/L hydrochloric acid conditioning solution pH value, stir 25min.Use 5mol/L sodium hydroxide solution regulation system pH value to 7.5 again, the ultraviolet lamp of wavelength 254nm is switched on power, put into retort internal radiation 20min, liquor capacity is 148L, 111L ethanol is slowly poured in retort, limit edged stirs, stir, precipitation 13h, remove supernatant liquor, 50L ethanol dehydration is added again in throw out, stir 2h and remove supernatant liquor, throw out adds 25L ethanol dehydration again, supernatant liquor is removed after 2h, throw out adds 12.5L ethanol dehydration again, supernatant liquor is removed after 2h, wet solid after dehydration is loaded in Stainless Steel Disc, put into vacuum drying case 60-65 DEG C of dry 45h, degradation product is weighed and obtains 7.22kg low molecular sodium heparin crude product, yield 72.20%.HPLC molecular chain conformation: molecular weight is 5950, >8000 part is 20.00% (answering 10.0-30.0%), <3000 part 9.5% (Ying≤30.0%), N-NO remains <0.25ppm (Ying≤0.25ppm).
Weighing 3kg sodium-chlor dissolves in purified water, and stirring and dissolving makes final volume be 150L, obtains the sodium chloride solution of 2%, weigh the low molecular sodium heparin crude product 0.4kg after degraded, add the sodium chloride solution of 2%, stirring and dissolving makes final volume be 3200mL, and regulator solution pH is 7.0, open constant flow pump, with the sodium chloride solution balance chromatography column 1-2h of 2%, equilibrium velocity is 140mL/min, balances complete, when sodium chloride solution trickling is to 1-2cm place in gel interface, close constant flow pump and outlet valve.Slowly added in chromatography column by solution constant flow pump after dissolving, first added-time flow velocity can not be too fast, prevents from destroying the smooth of gel interface.When solution adds to about 5cm on gel interface, open outlet valve, accelerate the flow velocity of application of sample simultaneously, until sample adds.When the trickling of low molecular sodium heparin crude product solution is in gel interface during 1-2cm place, start to connect sample.Carefully slowly pump into 2% sodium chloride solution with constant flow pump simultaneously.Receive 60 glasss of solution altogether, cumulative volume 30L, according to HPLC molecular chain conformation, the total area 64.70, macromolecule fraction is about 1.579, small molecules area about 9.977.The macromolecule fraction area being calculated removing by formula one is 2.44%, volume is 4.5L, the small molecules area that formula two calculates removing is 15.42%, volume is 8.5L, such intermediate collection area about 53.147, account for the total area 82.14%, volume 17.00L, therefore need to remove first 9 glasss and latter 17 glasss.Wash to open up in liquid to the middle 17.00L collected and add 34L ethanol, limit edged stirs, stir, precipitation leaves standstill 15h, remove supernatant liquor, 36.1L ethanol dehydration is added again in throw out, stir 2h and remove supernatant liquor, throw out adds 18.05L ethanol dehydration again, removes supernatant liquor after 2h, throw out adds 9.03L ethanol dehydration again, remove supernatant liquor after 2h, the wet solid after dehydration is loaded in Stainless Steel Disc, puts into vacuum drying case 60 ~ 65 DEG C of dry 48h, weigh and obtain low molecular sodium heparin fine work 0.298kg, yield 74.50%.
With above-mentioned chromatography by the low molecular sodium heparin purifying crude of other 6.82kg out, obtain low molecular sodium heparin fine work 5.53kg altogether, yield 76.59%.Low molecular sodium heparin fine work test molecule amount after being combined and distribution, meet the requirements.By low molecular sodium heparin fine work 5.53kg, 0.83kg sodium-chlor and 55.3L purified water add in 13# oxidation tank, stirring and dissolving.
Liquor capacity is 58.48L, with 5mol/L sodium hydrate regulator solution pH11.0.Add 263mL30% hydrogen peroxide, oxidization time 4.6h, test per hour pH value, maintenance system pH value 10.5-11.0.With 4mol/L hydrochloric acid conditioning solution pH8.1, add the reduction of 78.90g sodium sulphite anhydrous 99.3 and stir 30min.
By the solution ph to 5.8 that 4mol/L hydrochloric acid adjusts above-mentioned reaction complete, rinse 0.45um filter membrane to neutral by purified water, then itself and 13# oxidation tank are exported correct installation.Integrity test is carried out to 0.2 μm of filter membrane, detect qualified after be arranged on fixed position between 0.45um filter membrane and clean area setting tank, then start to filter, filtrate is placed in clean setting tank, the sodium chloride solution having filtered rear 11.70L1% rinses oxidation tank, washing fluid also puts into setting tank, and obtaining dalteparin sodium crude product solution cumulative volume is 66.4L.
Open whipping appts in setting tank, 132.8L ethanol is slowly added in setting tank, ethanol adds rear continuation and stirs 3-5min, then 10h is staticly settled, incline supernatant liquor, add 27.65L ethanol and stir dehydration to fine particulate, leave standstill 2h vacuum and pump supernatant liquor, add 13.83L ethanol and stir dehydration to fine particulate, leave standstill 2h vacuum and pump supernatant liquor, add 6.91L ethanol and stir dehydration to fine particulate, leave standstill 2h vacuum and pump supernatant liquor, to be dispensed in Stainless Steel Disc after dalteparin sodium throw out centrifuge dewatering 15min, a vacuum-drying 45-75 DEG C 40-44h obtains 4.28kg dalteparin sodium fine work, yield 77.40%.
Test example 1
By European Pharmacopoeia, every pharmacy index that embodiment 1-3 obtains dalteparin sodium fine work is tested, by assay record in table 1 below:
Table 1
In prior art the anti-Xa of dalteparin sodium fine work tire and the anti-Xa ratio that II a tires of tiring/resist all not high, anti-thrombus activity is not also very good (anti-Xa tires general below 150, and the anti-Xa ratio that II a tires of tiring/resist generally is no more than 3.0).As can be seen from Table 1, the dalteparin sodium obtained by the production method of dalteparin sodium fine work provided by the invention to be tired and anti-Xa tires/resists in the performance index such as ratio that II a tires and is obviously better than prior art at anti-Xa.
Above the production method of a kind of dalteparin sodium fine work provided by the present invention is described in detail.Apply specific case herein to set forth principle of the present invention and embodiment, the explanation of above embodiment just understands core concept of the present invention for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, some improvement and modification can also be carried out to the present invention.These improve and modify and also should fall in the protection domain of the claims in the present invention.
Claims (1)
1. a production method for dalteparin sodium fine work, is characterized in that comprising the steps:
(1) heparin sodium crude purified water is dissolved, regulator solution pH to 2.6-2.8, add Sodium Nitrite and stir regulator solution pH to 9.5-10.0 after 20min, obtain degradation solution, in degradation solution, add sodium borohydride stirring >=15h, regulator solution pH to 3.4-3.6 stirs 20min, ultra violet lamp degradation solution, with alcohol settling dehydration after regulator solution pH to 7.0-7.5, vacuum-drying obtains low molecular sodium heparin crude product; Described heparin sodium crude and purified water feed ratio are that every 1.0kg heparin sodium crude joins 9.2-9.5L purified water; The feed ratio of described heparin sodium crude and Sodium Nitrite is that every 100kg heparin sodium crude joins 1.5-2.0kg Sodium Nitrite; The feed ratio of described Sodium Nitrite and sodium borohydride is that 0.35-0.42kg sodium borohydride joined by every 1.0kg Sodium Nitrite; During Sodium Nitrite reaction, system temperature maintains 15-30 DEG C; By determining with starch-kalium iodide test paper test soln whether reaction terminates, and the solution of regulation system pH is: 4mol/L hydrochloric acid, 5mol/L sodium hydroxide in step (1); Ultra violet lamp wavelength is 254nm, the ratio adding liquor capacity and ethanol contend is 1.0:0.65-0.75, the Low molecular heparin crude product be settled out needs to use ethanol dehydration again three times in production, the ethanol of first time and the ratio of heparin sodium crude fed intake are the heparin sodium crude that every 3.0-5.0L ethanol is joined 1.0kg and fed intake, the ethanol of second time and third time is 50% of front ampoule, each dehydration interval more than 2h;
(2) above-mentioned low molecular sodium heparin crude product 2% sodium chloride solution is dissolved, the ratio of final solution volume and low molecular sodium heparin crude product weight is made to be that every 7.0-8.5L solution joins 1.0kg low molecular sodium heparin crude product, then add in chromatography column, with 2% sodium chloride solution at the uniform velocity wash-out, flow velocity is about column volume/2 hour, to the elutriant after chromatography by HPLC detection molecules amount and distribution, by formulae discovery remove large, small molecules per-cent number and corresponding volume, collect satisfactory elutriant and regulate pH to 7.0-7.5, the median elution liquid collected precipitates with the ethanol of its 2 times of volumes, throw out dewaters three times with ethanol again, the ethanol of first time is that 1.0kg low molecular sodium heparin crude product joined by every 3.0-5.0L ethanol with the ratio of the low molecular sodium heparin crude product fed intake, the ethanol of second time and third time is 50% of front ampoule, each dehydration interval more than 2h, vacuum-drying obtains low molecular sodium heparin fine work,
(3) the low molecular sodium heparin fine work gone out by gradation chromatography merges, test its molecular chain conformation, if small molecules part has deviation, regulator solution pH to 7.0-7.5, add alcohol settling to regulate small molecule segment, make it reach to meet the requirements, qualified low molecular sodium heparin fine work is added in 13# oxidation tank, add purified water and sodium-chlor, the ratio of low molecular sodium heparin fine work and purified water is that 8.5-10L purified water joined by every 1.0kg low molecular sodium heparin fine work, and the ratio of sodium-chlor and purified water is that every 0.8-1.5kg sodium-chlor joins 100L purified water, stirring and dissolving, step (3) if in merge after low molecular sodium heparin fine work HPLC detection molecules amount and the distribution display molecular weight small molecules part that is less than 3000 do not meet≤13.0% requirement, the low molecular sodium heparin fine work repurity one time just will merged, be dissolved in purified water, low molecular sodium heparin fine work and purified water feed ratio are that 8.5-10L purified water joined by every 1.0kg low molecular sodium heparin fine work, regulate pH7.0-7.5, precipitate with the ethanol of 2 times of volumes again, throw out dewaters three times with ethanol again, the ethanol of first time is that 1.0kg low molecular sodium heparin fine work joined by every 3.0-5.0L ethanol with the ratio of the low molecular sodium heparin fine work fed intake, the ethanol of second time and third time is 50% of front ampoule, each dehydration interval more than 2h,
(4) regulate low molecular sodium heparin fine work pH value of solution to 10.5-11.0, add 30% hydrogen peroxide oxidation >=4-5h, rear adjustment pH to 7.9-8.1, adds sodium sulphite anhydrous 99.3, stirs 30min filtration and obtains dalteparin sodium crude product solution; The ratio of 30% hydrogen peroxide and liquor capacity is 0.25-0.45:100, and the ratio of sodium sulphite anhydrous 99.3 and hydrogen peroxide is that 0.20-0.30kg sodium sulphite anhydrous 99.3 joins 1.0L hydrogen peroxide;
(5) regulate dalteparin sodium crude product solution pH5.6-5.8, make its by 0.45 μm and 0.2 μm of filter membrane degerming, the dalteparin sodium solution collection after Sterile Filtration is in clean setting tank; Need in step (5) to do 0.2 μm and 0.45 μm of integrity of filtration membranes test, to guarantee that the filter membrane used in production can meet Sterile Filtration requirement;
(6) alcohol settling of 2.0 times of volumes of the dalteparin sodium solution after degerming, the DALT fine work ethanol dehydration be settled out three times, the ethanol of first time is that 1.0kg low molecular sodium heparin fine work joined by every 3.0-5.0L ethanol with the ratio of the low molecular sodium heparin fine work fed intake, the ethanol of second time and third time is 50% of front ampoule, each dehydration interval more than 2h; Last vacuum-drying obtains fine work dalteparin sodium.
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| CN105884934A (en) * | 2014-10-17 | 2016-08-24 | 北京海吉星医疗科技有限公司 | Preparation method of dalteparin sodium |
| CN104403025B (en) * | 2014-12-24 | 2016-10-05 | 南京健友生化制药股份有限公司 | A kind of liquaemin removes color method |
| CN105294885A (en) * | 2015-11-23 | 2016-02-03 | 山东大学 | Preparation method of novel source of low molecular weight heparin from nitrous acid degradation |
| CN107236057B (en) * | 2017-05-19 | 2019-08-06 | 南京健友生化制药股份有限公司 | A kind of biodegrading process obtaining Dalteparin Sodium |
| EP3740513B1 (en) | 2018-02-14 | 2022-06-22 | Biological E Limited | Improved process for the preparation of dalteparin sodium |
| CN114276473A (en) * | 2020-09-27 | 2022-04-05 | 上海帕尼生物科技有限公司 | Refined heparin sodium product and extraction method and application thereof |
| CN113024689B (en) * | 2021-05-21 | 2021-08-13 | 江西浩然生物制药有限公司 | Method for controlling molecular weight of dalteparin sodium |
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