CN104098716A - 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 of pig or ox, belongs to mucopolysaccharide material.All having in vivo and in vitro blood coagulation resisting function, is the anticoagulation medicine of current main flow.But the bioavailability of unfractionated heparin sodium is low, and side effect is large, it is found that afterwards the sodium salt of the CSSO3 fragment that heparin sodium cracking is obtained, molecular-weight average is 4000～6000 dalton, is called low molecular sodium heparin.Low molecular heparin sodium injection belong to antithrombin Ⅲ (AT III) dependency thrombin inhibitors the same as heparin sodium injection.But compare with heparin sodium injection, have that the transformation period is long, antithrombotic effect is good, bleeding tendency is weak, convenient drug administration, but price comparison is expensive.And DALT is for wherein a kind of.Dalteparin sodium injection liquid is mainly used in the treatment of acute deep venous thrombosis, acute renal failure or chronic renal insufficiency person carry out preventing during hemodialysis and blood filtration occurring in extracorporeal circulation system blood coagulation, instability mode coronary heart disease, as unstable angina pectoris and non-Q-wave mode myocardial infarction, the thrombosis that prevention is relevant with operation.It is the antithrombotic class medicine of current main flow.
At present existing 3 pieces of patent reports preparation process of dalteparin sodium, wherein have 2 pieces to authorize.Patent CN101942038A by by heparin sodium material dissolution in purified water and the organic solvent that contains methyl, under certain temperature and pH value condition, add Sodium Nitrite to degrade, after under certain pH value condition, with sodium hydroxide, reduce again, then with ethanol precipitation, membrane filtration, ultraviolet disinfection, hydrogen peroxide oxidation membrane filtration, then by ethanol precipitation, throw out lyophilize or dewatering and vacuum drying are obtained to dalteparin sodium finished product.Its Patent CN102558393A adopts the preparation technology of the most traditional dalteparin sodium, first heparin sodium is dissolved in and in purified water, obtains the heparin sodium aqueous solution, the pH value of hydrochloric acid regulating solution, afterwards with the Sodium Nitrite heparin sodium of degrading, again by sodium hydroxide regulation system pH value, sodium borohydride reduction, precipitates and obtains precipitate reduction thing with ethanol afterwards, again it is obtained to crude product dalteparin sodium with hydrogen peroxide oxidation, then after carrying out ultrafiltration with ultra-filtration membrane, freeze-drying obtains fine work dalteparin sodium finished product.
Above-mentioned 2 pieces of patent basic ideas of having authorized are consistent, but all have certain disadvantages, and patent CN101942038A be take refined heparin sodium as raw material in preparation process, are difficult to control from source the quality of heparin sodium, and have increased the cost of preparing dalteparin sodium.In preparation process, added the organic solvent that contains methyl, cause in final dalteparin sodium raw material standard and need to control organic solvent residual, increase testing amount and the quality that has reduced product, with lack degradation condition system, accurate control, thereby cause the Low molecular heparin purity for preparing inadequate, molecular weight distribution is not concentrated, the residual too much shortcoming of impurity, these all current technique cann't be solved.There is certain shortcoming in the preparation of supplying with injection medicine.Patent CN102558393A has carried out certain simplification on the basis of above-mentioned that patent CN101942038A, first cancelled the use of the organic solvent that contains methyl, reduced the consumption of purified water, after having reduced the operation steps of filtration and ultraviolet degerming and hydrogen peroxide oxidation, must filter, directly use molecular cut-off membrane filtration, obtain dalteparin sodium fine work.This patent be take refined heparin sodium equally as raw material, and on producing, obtaining of raw material is subject to certain restrictions like this, and increased cost, is difficult to control from source production technique and the quality product of whole dalteparin sodium simultaneously.Used expensive molecular cut-off filtering membrane, molecular cut-off filtering membrane is used frequent, has increased 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 is used heparin sodium crude can obtain the good dalteparin sodium fine work of quality and obtain higher yield for raw material, has realized that dalteparin sodium is low-cost, high efficiency suitability for industrialized production.
A kind of technique of 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 to dissolve.Regulator solution pH to 2.6-2.8, adds Sodium Nitrite, and maintain the temperature at 15-25 ℃ and stir 20min, then regulator solution pH to 9.5-10.0, adding 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 is penetrated degradation solution 20-25min, adds ethanol precipitation, and more than standing 8h, the solid after precipitation is put into vacuum drying oven after dewatering again, and 60-65 ℃ of dry 40-48h, obtains low molecular sodium heparin crude product.
(2) low molecular sodium heparin fine work preparation (gel filtration chromatography)
After above-mentioned low molecular sodium heparin crude product is dissolved with 2% sodium chloride solution, add in chromatography column, with 2% sodium chloride solution, at the uniform velocity wash and open up, receive solution, measure the ultraviolet OD200 absorbance value of each glass, according to numerical value, draw collection of illustrative plates.By molecular weight and the distributed data of HPLC test, determine the large and small molecule per-cent number that need remove by gel chromatography, by formula, calculate large and small molecular area and the corresponding volume that corresponding need are removed.Collect the solution part needing and regulate pH to 7.0-7.5, the standing >=8h of ethanol precipitation, wet solid 60-65 ℃ of vacuum-drying 18-24h after dehydration.
(3) mixed dissolution
The low molecular sodium heparin fine work that chromatography goes out is in batches merged, test its molecular weight and distribution, if small molecules partly has deviation, adjustable pH value of solution 7.0-7.5, adds ethanol precipitation to regulate small molecule segment, it is reached and meet the requirements.By molecular weight and distribute low molecular sodium heparin fine work qualified and add in 13# oxidation tank, add 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.After finishing, regulate pH7.9-8.1, filter after adding sodium sulphite anhydrous 99.3 to stir 30min, obtain dalteparin sodium crude product solution.
(5) Sterile Filtration
Regulate dalteparin sodium crude product solution pH5.6-5.8, by 0.45 μ m and 0.2 μ m filter membrane series winding, outlet is connected 0.45 μ m port with 13# oxidation tank, open retort outlet valve, make pot liquid successively by 0.45 μ m and 0.2 μ m filtering membrane, the dalteparin sodium solution after Sterile Filtration is collected in clean setting tank.
(6) preparation of dalteparin sodium fine work
Dalteparin sodium solution after filtration add ethanol precipitation standing >=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 of heparin sodium crude and Sodium Nitrite (W/W) is 100:1.5-2.5, is preferably 100:1.5-2.0.The feed ratio of Sodium Nitrite and sodium borohydride (W/W) is 1.0:0.3-0.5, is preferably 1.0:0.35-0.42.During Sodium Nitrite reaction, system temperature maintains 15-30 ℃, and 4mol/L hydrochloric acid regulates pH2.6-2.8, by determining that with starch-kalium iodide test paper test soln reaction finishes, 5mol/L sodium hydrate regulator solution pH to 9.0-10.5.The solution that 254nm ultraviolet lamp is placed in after degraded carries out sterilizing, and adding liquor capacity and ethanol volume (V/V) is 1.0:0.65-0.75, and preferably 0.65-0.70 precipitates.The low molecular sodium heparin crude product being settled out needs to use ethanol dehydration three times on producing again, the ethanol of use is 3.0-5.0:1.0 with the ratio (V/W) of the heparin sodium crude feeding intake for the first time, be preferably 3.5-4.0:1.0, for the second time and for the third time the ethanol of use is front ampoule 50%, more than each dehydration interval 2h, and the Low molecular heparin sodium sample obtaining is carried out to molecular weight and distribution tests.
In step (2), need 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) for 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, guarantees the smooth of gel interface in application of sample process.Add after sample, with 2% sodium chloride solution, at the uniform velocity wash and open up gel column, the HPLC molecular weight by crude product above and distribution tests result also calculate by formula below large and small molecular area and the corresponding volume that corresponding need are removed.
A. the per-cent of establishing the macromole part area of removing is X%
the macromole content %-X% of low molecular raw material=(15～25) %
100％-X％
X%=
macromole content %-(15～25) % of low molecular raw material
100％-(15～25)％
＝____________＝____________＝__________％
Formula one
B. the per-cent of establishing the small molecules part area of removing is Y%
the small molecules content %-Y% of low molecular raw material=-≤13%
100％-Y％
Y%=
small molecules content %+≤13% of low molecular raw material
100％+≤13％
＝__________＝____________＝__________％
Formula two
The centre of collecting is washed and is opened up liquid and precipitate with the ethanol of its 2 times of volumes, throw out dewaters three times with ethanol again, the ethanol of use is 3.0-5.0:1.0 with the ratio (V/W) of the low molecular sodium heparin crude product feeding intake for the first time, be preferably 3.5-4.0:1.0, for the second time and for the third time the ethanol of use is front ampoule 50%, dewaters more than interval 2h at every turn.
In step (3), be mainly that the low molecular sodium heparin that chromatography is gone out carries out the test of molecular weight and molecualr weight distribution, if the result testing out shows small molecules part (<3000) and does not meet≤13.0% requirement, can by purified water, dissolve chromatography low molecular sodium heparin fine work out, and regulate pH7.0-7.5, ethanol precipitation, reaches it and meets the requirements.By molecular weight and the low molecular sodium heparin fine work that distributes qualified and purified water (W/V), be 1.0:8.5-10, 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.
In step (4), by the solution sodium hydrate regulator solution pH10.5-11.0 in above-mentioned steps (3), 30% hydrogen peroxide adding and solution (V/V) are 0.25-0.45:100, are preferably 0.25-0.3:100, and 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.
In step (5), need to do 0.2 μ m and the test of 0.45 μ m integrity of filtration membranes.Guarantee that the filter membrane using in producing can meet Sterile Filtration requirement.
The ethanol precipitation of 2.0 times of volumes for dalteparin sodium solution after filtering in step (6).Ethanol dehydration three times for the dalteparin sodium fine work being settled out, the ethanol of use is 3.0-5.0:1.0 with the ratio (V/W) of the low molecular sodium heparin fine work feeding intake for the first time, be preferably 3.5-4.0:1.0, for the second time and for the third time the ethanol of use is front ampoule 50%, dewaters more than interval 2h at every turn.
The present invention be directed to existing dalteparin sodium production technique is optimized and improves, use heparin sodium crude is raw material, now by degraded, reduction, ethanol precipitation, obtain low molecular sodium heparin crude product, with respect to needs, use refined heparin sodium for raw material, the method scope of application is wider, more can control from source the quality of dalteparin sodium fine work.The passing through of novelty tests to the HPLC of the low molecular sodium heparin crude product before chromatography the collection that its molecular weight and distribution and formula calculate filtrate in chromatography process, determine to wash and open up that in liquid, which needs to retain, which need to be given up, and very effectively and accurately quantitatively arrive satisfactory low molecular sodium heparin fine work liquid, avoided 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 filtering, precipitate, being dried.From oxidation, Sterile Filtration, precipitation dehydration, realized the technique of " treating different things alike ", in operation, realized simplification, time and human cost have been saved, this technique has been developed a new method of preparing dalteparin sodium fine work in existing technical foundation, and Qie our company has realized the suitability for industrialized production of dalteparin sodium.
Embodiment
In order to make those skilled in the art person understand better technical scheme of the present invention, below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
Add 90L water and heparin sodium crude 10kg to retort, stirring is dissolved it completely, with solution in 4mol/L hydrochloric acid regulating tank, be that pH value is 2.7, take 0.18kg Sodium Nitrite and add in beaker, in beaker, add 1620mL purified water, after stirring and dissolving, add in retort, maintaining pH in tank is 2.6-2.8, uses starch-kalium iodide test paper test soln after stirring reaction 20min, observe test paper color, until the not aobvious blueness of test paper.By 5mol/L sodium hydroxide solution regulation system pH value, be 9.6, add 0.072kg sodium borohydride to retort, stirring reaction 16h, then be 3.5 by 4mol/L hydrochloric acid conditioning solution pH value, stir 20min.Again by 5mol/L sodium hydroxide solution regulation system pH value to 7.5, 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, precipitate standing 10h, remove supernatant liquor, in throw out, add again 40L ethanol dehydration, stir 2h and remove supernatant liquor, throw out adds 20L ethanol dehydration again, after 2h, remove supernatant liquor, throw out adds 20L ethanol dehydration again, after 2h, remove supernatant liquor, wet solid after dehydration is packed in Stainless Steel Disc, put into 60～65 ℃ of dry 48h of vacuum drying case, degradation product is weighed and obtained 7.15kg low molecular sodium heparin crude product, yield 71.50%.HPLC molecular weight and distribution: molecular weight is 6550, >8000 is partly 26.51% (answering 10.0-30.0%), <3000 part 15.5% (Ying≤30.0%), the residual <0.25ppm (of N-NO Ying≤0.25ppm).
Weigh 3kg sodium-chlor and dissolve in purified water, it is 150L that stirring and dissolving makes final volume, obtains 2% sodium chloride solution, weigh the low molecular sodium heparin crude product 0.4kg after degraded, add 2% sodium chloride solution, it is 3200mL that stirring and dissolving makes final volume, and regulator solution pH is 7.0, open constant flow pump, with 2% sodium chloride solution balance chromatography column 1-2h, equilibrium velocity is 145mL/min, and balance is complete, when sodium chloride solution trickles to 1-2cm place in gel interface, close constant flow pump and outlet valve.Solution after dissolving is slowly added in chromatography column with constant flow pump, and first added-time flow velocity can not be too fast, prevents from destroying the smooth of gel interface.On solution adds to gel interface, during 5cm left and right, open outlet valve, accelerate the flow velocity of application of sample, until sample adds simultaneously.When low molecular sodium heparin crude product solution, trickle in gel interface during 1-2cm place, start to connect sample.Meanwhile, with constant flow pump, carefully slowly pump into 2% sodium chloride solution.Receive altogether 60 glasss of solution, cumulative volume 30L, according to HPLC molecular weight and distribution, the total area 65.29, macromole is partly about 6.79, small molecules part area approximately 13.71.By formula one, calculating the macromole part area of removing is 10.4%, volume is 6.5L, it is 21% that formula two calculates the small molecules part area of removing, volume is 10.75L, intermediate collection part area approximately 44.79 like this, account for the total area 68.6%, volume 12.75L, therefore need remove first 13 glasss and latter 21.5 glasss, to the middle 12.75L collecting, wash to open up and in liquid, add 25.5L ethanol, limit edged stirs, stir, precipitate standing 14h, remove supernatant liquor, in throw out, add again 28.6L ethanol dehydration, stir 2h and remove supernatant liquor, throw out adds 14.3L ethanol dehydration again, after 2h, remove supernatant liquor, throw out adds 7.2L ethanol dehydration again, after 2h, remove supernatant liquor, wet solid after dehydration is packed in Stainless Steel Disc, put into 60-65 ℃ of dry 48h of vacuum drying case, weigh and obtain low molecular sodium heparin fine work 0.31kg, yield 77.50%.
By above-mentioned chromatography by the low molecular sodium heparin purifying crude of other 6.75kg out, obtain altogether low molecular sodium heparin fine work 5.41kg, yield 75.66%.Low molecular sodium heparin fine work test molecule amount and distribution after being combined, 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, pH value of test per hour, 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.
With 4mol/L hydrochloric acid, regulate the complete pH to 5.8 of above-mentioned reaction, by purified water, rinse 0.45um filter membrane to neutral, then itself and 13# oxidation tank are exported to correct installation.0.2 μ m filter membrane is carried out to integrity test, detect after qualified and 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, sodium-chlor flushing oxidation tank with 10.4L1% after filtration completes is also put 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 staticly settle 14h, supernatant liquor inclines, adding 21.64L ethanol to stir dewaters to fine particulate, standing 2h, vacuum pumps supernatant liquor, adding 10.82L ethanol to stir dewaters to fine particulate, standing 2h vacuum pumps supernatant liquor, adding 5.41L ethanol to stir dewaters to fine particulate, standing 2h vacuum pumps supernatant liquor, dalteparin sodium throw out was installed in Stainless Steel Disc with after centrifuge dewatering 15min minute, a vacuum-drying 45-75 ℃ 40-44h obtains 4.12kg dalteparin sodium fine work, yield 76.16%.
Embodiment 2
Add 85L water and heparin sodium crude 10kg to retort, stirring is dissolved it completely, with solution in 4mol/L hydrochloric acid regulating tank, be that pH value is 2.7, take 0.15kg Sodium Nitrite and add in beaker, in beaker, add 1650mL purified water, after stirring and dissolving, add in retort, maintaining pH in tank is 2.6-2.8, uses starch-kalium iodide test paper test soln after stirring reaction 25min, observe test paper color, until the not aobvious blueness of test paper.By 5mol/L sodium hydroxide solution regulation system pH value, be 9.8, add 0.045kg sodium borohydride to retort, stirring reaction 18h, then be 3.5 by 4mol/L hydrochloric acid conditioning solution pH value, stir 23min.Again by 5mol/L sodium hydroxide solution regulation system pH value to 7.3, 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, in throw out, add again 30L ethanol dehydration, stir 2h and remove supernatant liquor, throw out adds 15L ethanol dehydration again, after 2h, remove supernatant liquor, throw out adds 7.5L ethanol dehydration again, after 2h, remove supernatant liquor, wet solid after dehydration is packed in Stainless Steel Disc, put into 60-65 ℃ of dry 44h of vacuum drying case, degradation product is weighed and obtained 7.18kg low molecular sodium heparin crude product, yield 71.80%.HPLC molecular weight and distribution: molecular weight is 6450, >8000 is partly 26.50% (answering 10.0-30.0%), <3000 part 15.5% (Ying≤30.0%), the residual <0.25ppm (of N-NO Ying≤0.25ppm).
Weigh 3kg sodium-chlor and dissolve in purified water, it is 150L that stirring and dissolving makes final volume, obtains 2% sodium chloride solution, weigh the low molecular sodium heparin crude product 0.4kg after degraded, add 2% sodium chloride solution, it is 3200mL that stirring and dissolving makes final volume, and regulator solution pH is 7.0, open constant flow pump, with 2% sodium chloride solution balance chromatography column 1-2h, equilibrium velocity is 140mL/min, and balance is complete, when sodium chloride solution trickles to 1-2cm place in gel interface, close constant flow pump and outlet valve.Solution after dissolving is slowly added in chromatography column with constant flow pump, and first added-time flow velocity can not be too fast, prevents from destroying the smooth of gel interface.On solution adds to gel interface, during 5cm left and right, open outlet valve, accelerate the flow velocity of application of sample, until sample adds simultaneously.When low molecular sodium heparin crude product solution, trickle in gel interface during 1-2cm place, start to connect sample.With constant flow pump, carefully slowly pump into 2% sodium chloride solution simultaneously.Receive altogether 60 glasss of solution, cumulative volume 30L, according to HPLC molecular weight and distribution, the total area 67.74, macromole is partly about 7.04, small molecules part area approximately 14.23.By formula one, calculating the macromole part area of removing is 10.4%, volume is 7.75L, it is 21% that formula two calculates the small molecules part area of removing, volume is 9.5L, intermediate collection part area approximately 46.47 like this, account for the total area 68.6%, volume 12.75L, therefore need remove first 15.5 glasss and latter 19 glasss.To the middle 12.75L collecting, wash to open up and in liquid, add 25.5L ethanol, limit edged stirs, stir the standing 14h of precipitation and remove supernatant liquor, in throw out, add again 28.6L ethanol dehydration, stir 2h and remove supernatant liquor, throw out adds 14.3L ethanol dehydration again, after 2h, remove supernatant liquor, throw out adds 7.2L ethanol dehydration again, after 2h, remove supernatant liquor, the wet solid after dehydration is packed in Stainless Steel Disc, put into 60-65 ℃ of dry 48h of vacuum drying case, weigh and obtain low molecular sodium heparin fine work 0.29kg, yield 72.50%.
By above-mentioned chromatography by the low molecular sodium heparin purifying crude of other 6.78kg out, obtain altogether low molecular sodium heparin fine work 5.45kg, yield 75.91%.Low molecular sodium heparin fine work test molecule amount and distribution after being combined, 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, pH value of test per hour, 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.
With 4mol/L hydrochloric acid, regulate the complete pH to 5.6 of above-mentioned reaction, by purified water, rinse 0.45um filter membrane to neutral, then itself and 13# oxidation tank are exported to correct installation.0.2 μ m filter membrane is carried out to integrity test, detect after qualified and 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, sodium chloride solution with 9.85L1% after filtration completes rinses oxidation tank, washing fluid is also put 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 staticly settle 10h, supernatant liquor inclines, adding 16.35L ethanol to stir dewaters to fine particulate, standing 2h, vacuum pumps supernatant liquor, adding 8.18L ethanol to stir dewaters to fine particulate, standing 2h, vacuum pumps supernatant liquor, adding 4.09L ethanol to stir dewaters to fine particulate, standing 2h vacuum pumps supernatant liquor, dalteparin sodium throw out was installed in Stainless Steel Disc with after centrifuge dewatering 15min minute, a vacuum-drying 45-75 ℃ 40-44h obtains 4.14kg dalteparin sodium fine work, yield 75.96%.
Embodiment 3
Add 100L water and heparin sodium crude 10kg to retort, stirring is dissolved it completely, with pH in 4mol/L hydrochloric acid regulating tank, be 2.7, take 0.25kg Sodium Nitrite and add in beaker, in beaker, add 1550mL purified water, after stirring and dissolving, add in retort, maintaining pH in tank is 2.6-2.8, uses starch-kalium iodide test paper test soln after stirring reaction 25min, observe test paper color, until the not aobvious blueness of test paper.By 5mol/L sodium hydroxide solution regulation system pH value, be 10.0, add 0.125kg sodium borohydride to retort, stirring reaction 16h, then be 3.6 by 4mol/L hydrochloric acid conditioning solution pH value, stir 25min.Again by 5mol/L sodium hydroxide solution regulation system pH value to 7.5, 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, in throw out, add again 50L ethanol dehydration, stir 2h and remove supernatant liquor, throw out adds 25L ethanol dehydration again, after 2h, remove supernatant liquor, throw out adds 12.5L ethanol dehydration again, after 2h, remove supernatant liquor, wet solid after dehydration is packed in Stainless Steel Disc, put into 60-65 ℃ of dry 45h of vacuum drying case, degradation product is weighed and obtained 7.22kg low molecular sodium heparin crude product, yield 72.20%.HPLC molecular weight and distribution: molecular weight is 5950, >8000 is partly 20.00% (answering 10.0-30.0%), <3000 part 9.5% (Ying≤30.0%), the residual <0.25ppm (of N-NO Ying≤0.25ppm).
Weigh 3kg sodium-chlor and dissolve in purified water, it is 150L that stirring and dissolving makes final volume, obtains 2% sodium chloride solution, weigh the low molecular sodium heparin crude product 0.4kg after degraded, add 2% sodium chloride solution, it is 3200mL that stirring and dissolving makes final volume, and regulator solution pH is 7.0, open constant flow pump, with 2% sodium chloride solution balance chromatography column 1-2h, equilibrium velocity is 140mL/min, and balance is complete, when sodium chloride solution trickles to 1-2cm place in gel interface, close constant flow pump and outlet valve.Solution after dissolving is slowly added in chromatography column with constant flow pump, and first added-time flow velocity can not be too fast, prevents from destroying the smooth of gel interface.On solution adds to gel interface, during 5cm left and right, open outlet valve, accelerate the flow velocity of application of sample, until sample adds simultaneously.When low molecular sodium heparin crude product solution, trickle in gel interface during 1-2cm place, start to connect sample.With constant flow pump, carefully slowly pump into 2% sodium chloride solution simultaneously.Receive altogether 60 glasss of solution, cumulative volume 30L, according to HPLC molecular weight and distribution, the total area 64.70, macromole is partly about 1.579, small molecules part area approximately 9.977.By formula one, calculating the macromole part area of removing is 2.44%, volume is 4.5L, it is 15.42% that formula two calculates the small molecules part area of removing, volume is 8.5L, intermediate collection part area approximately 53.147 like this, account for the total area 82.14%, volume 17.00L, therefore need remove first 9 glasss and latter 17 glasss.To the middle 17.00L collecting, wash to open up and in liquid, add 34L ethanol, limit edged stirs, stir, precipitate standing 15h, remove supernatant liquor, in throw out, add again 36.1L ethanol dehydration, 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, after 2h, remove supernatant liquor, the wet solid after dehydration is packed in Stainless Steel Disc, put into 60～65 ℃ of dry 48h of vacuum drying case, weigh and obtain low molecular sodium heparin fine work 0.298kg, yield 74.50%.
By above-mentioned chromatography by the low molecular sodium heparin purifying crude of other 6.82kg out, obtain altogether low molecular sodium heparin fine work 5.53kg, yield 76.59%.Low molecular sodium heparin fine work test molecule amount and distribution after being combined, 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, pH value of test per hour, 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 to stir 30min.
With 4mol/L hydrochloric acid, adjust the complete pH to 5.8 of above-mentioned reaction, by purified water, rinse 0.45um filter membrane to neutral, then itself and 13# oxidation tank are exported to correct installation.0.2 μ m filter membrane is carried out to integrity test, detect after qualified and 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, sodium chloride solution with 11.70L1% after filtration completes rinses oxidation tank, washing fluid is also put 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 staticly settle 10h, supernatant liquor inclines, adding 27.65L ethanol to stir dewaters to fine particulate, standing 2h vacuum pumps supernatant liquor, adding 13.83L ethanol to stir dewaters to fine particulate, standing 2h vacuum pumps supernatant liquor, adding 6.91L ethanol to stir dewaters to fine particulate, standing 2h vacuum pumps supernatant liquor, dalteparin sodium throw out was installed in Stainless Steel Disc with after centrifuge dewatering 15min minute, a vacuum-drying 45-75 ℃ 40-44h obtains 4.28kg dalteparin sodium fine work, yield 77.40%.
Test example 1
The every pharmacy index that embodiment 1-3 is obtained to dalteparin sodium fine work by European Pharmacopoeia is tested, and assay is recorded in following table 1:
Table 1
In prior art the anti-Xa of dalteparin sodium fine work tire and tire/ratio that anti-II a tires of anti-Xa all not high, anti-thrombus activity is not also very good (anti-Xa tires generally below 150, anti-Xa tires/ratio that anti-II a tires is generally no more than 3.0).As can be seen from Table 1, the dalteparin sodium that the production method by dalteparin sodium fine work provided by the invention obtains anti-Xa tire and tire/performance index such as ratio that anti-II a tires of anti-Xa on be obviously better than prior art.
Above the production method of a kind of dalteparin sodium fine work provided by the present invention is described in detail.Applied specific case herein principle of the present invention and embodiment are set forth, the explanation of above embodiment is just for helping to understand core concept of the present invention.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention.These improvement and modification also should fall in the protection domain of the claims in the present invention.
Claims (8)
1. a production method for dalteparin sodium fine work, is characterized in that comprising the steps:
(1) heparin sodium crude dissolves by purified water, regulator solution pH to 2.6-2.8, add Sodium Nitrite to 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, after regulator solution pH to 7.0-7.5, with the dehydration of ethanol precipitation, vacuum-drying obtains low molecular sodium heparin crude product; Described heparin sodium crude and purified water feed ratio (W/V) are 1.0:8.5-10; The feed ratio of described heparin sodium crude and Sodium Nitrite (W/W) is 100:1.5-2.5; The feed ratio of described Sodium Nitrite and sodium borohydride (W/W) is 1.0:0.3-0.5;
(2) after being dissolved with 2% sodium chloride solution, above-mentioned low molecular sodium heparin crude product adds in chromatography column, with 2% sodium chloride solution, at the uniform velocity wash and open up, HPLC detection molecules amount and distribution, by formula, calculate large and small molecule per-cent number and the corresponding volume of removing, collecting satisfactory washing opens up liquid and regulates pH to 7.0-7.5, the dehydration of ethanol precipitation, vacuum-drying obtains low molecular sodium heparin fine work;
(3) low molecular sodium heparin fine work gradation chromatography being gone out merges, test its molecular weight and distribution, if small molecules partly has deviation, adjustable pH value of solution is to 7.0-7.5, add ethanol precipitation to regulate small molecule segment, it reached and meet the requirements, qualified low molecular sodium heparin fine work be added to 13# oxidation tank interior in, add purified water and sodium-chlor, stirring and dissolving;
(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 adjusting pH to 7.9-8.1, adds sodium sulphite anhydrous 99.3, stirs 30min filtration and obtains dalteparin sodium crude product solution;
(5) regulate dalteparin sodium crude product solution pH5.6-5.8, make it pass through 0.45 μ m and 0.2 μ m filter membrane degerming, the dalteparin sodium solution after Sterile Filtration is collected in clean setting tank;
(6) second alcohol precipitation ﹑ dehydration for the dalteparin sodium solution after degerming, vacuum-drying obtain fine work dalteparin sodium.
2. the production method of dalteparin sodium fine work according to claim 1, is characterized in that, in step (1), heparin sodium crude and purified water feed ratio (W/V) are 1.0:9.2-9.5; The feed ratio of heparin sodium crude and Sodium Nitrite (W/W) is 100:1.5-2.0; The feed ratio of Sodium Nitrite and sodium borohydride (W/W) is 1.0:0.35-0.42; During Sodium Nitrite reaction, system temperature maintains 15-30 ℃, pH2.6-2.8.
3. the production method of dalteparin sodium fine work according to claim 1, it is characterized in that, in step (1) by determining with starch-kalium iodide test paper test soln whether reaction finishes, and the solution of regulation system pH is: 4mol/L hydrochloric acid, 5mol/L sodium hydroxide; Ultra violet lamp wavelength is 254nm, the ratio (V/V) that adds liquor capacity and ethanol volume is 1.0:0.65-0.75, the Low molecular heparin crude product being settled out needs to use ethanol dehydration three times on producing again, the ethanol of use is 3.0-5.0:1.0 with the ratio (V/W) of the heparin sodium crude feeding intake for the first time, for the second time and for the third time the ethanol of use is front ampoule 50%, dewaters more than interval 2h at every turn.
4. the production method of dalteparin sodium fine work according to claim 1, it is characterized in that, the low molecular sodium heparin crude product that step (2) obtains with 2% sodium chloride solution dissolving step (1), make final solution volume and low molecular sodium heparin crude product weight (V/W) for 7.0-8.5:1.0, with 2% sodium chloride solution, wash to open up to wash again and open up chromatography column, flow velocity is about column volume/2 hour, washing after chromatography opened up to liquid and by HPLC, test its molecular weight and distribution, according to formula, calculate large that corresponding need remove again, small molecules area and corresponding volume, the centre of collecting is washed and is opened up liquid and precipitate with the ethanol of its 2 times of volumes, throw out dewaters three times with ethanol again, the ethanol of use is 3.0-5.0:1.0 with the ratio (V/W) of the low molecular sodium heparin crude product feeding intake for the first time, for the second time and for the third time the ethanol of use is front ampoule 50%, more than each dehydration interval 2h.
5. the production method of dalteparin sodium fine work according to claim 1, it is characterized in that step (3) if in low molecular sodium heparin fine work HPLC detection molecules amount after merging and distribute and show that small molecules part (<3000) does not meet requirement of≤13.0%, can be by the low molecular sodium heparin fine work repurity merging one time, be dissolved in purified water, low molecular sodium heparin fine work and purified water feed ratio (W/V) are 1.0:8.5-10, regulate pH7.0-7.5, with the ethanol of 2 times of volumes, precipitate again, throw out dewaters three times with ethanol again, the ethanol of use is 3.0-5.0:1.0 with the ratio (V/W) of the low molecular sodium heparin fine work feeding intake for the first time, for the second time and for the third time the ethanol of use is front ampoule 50%, more than each dehydration interval 2h, during qualified low molecular sodium heparin fine work is added in 13# oxidation tank, the ratio (W/V) of low molecular sodium heparin fine work and purified water is 1.0:8.5-10, the ratio (W/V) of sodium-chlor and purified water is 0.8-1.5:100.
6. the production method of dalteparin sodium fine work according to claim 1, it is characterized in that, in step (4), the solution in above-mentioned steps (3) is regulated to pH to 10.5-11.0 with sodium hydroxide, the ratio (V/V) of 30% hydrogen peroxide and solution is 0.25-0.45:100, and sodium sulphite anhydrous 99.3 and hydrogen peroxide (W/V) are 0.20-0.30:1.0.
7. the production method of dalteparin sodium fine work according to claim 1, is characterized in that needing to do 0.2 μ m and the test of 0.45 μ m integrity of filtration membranes in step (5), to guarantee that the filter membrane using in production can meet Sterile Filtration requirement.
8. the production method of dalteparin sodium fine work according to claim 1, it is characterized in that the dalteparin sodium solution after filtering in step (6) precipitates with the ethanol of 2.0 times of volumes, ethanol dehydration three times for the DALT fine work being settled out, the ethanol of use is 3.0-5.0:1.0 with the ratio (V/W) of the low molecular sodium heparin fine work feeding intake for the first time, for the second time and for the third time the ethanol of use is front ampoule 50%, dewaters more than interval 2h at every turn.
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