CN103421128A - Method for preparing high-quality low molecular parnaparin sodium - Google Patents
Method for preparing high-quality low molecular parnaparin sodium Download PDFInfo
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- 229920000669 heparin Polymers 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 56
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 46
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 30
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims abstract description 25
- 229960001008 heparin sodium Drugs 0.000 claims abstract description 25
- 238000002360 preparation method Methods 0.000 claims abstract description 23
- 239000012528 membrane Substances 0.000 claims abstract description 19
- 230000002745 absorbent Effects 0.000 claims abstract description 17
- 239000002250 absorbent Substances 0.000 claims abstract description 17
- 238000001035 drying Methods 0.000 claims abstract description 9
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 claims abstract description 7
- 239000002994 raw material Substances 0.000 claims abstract description 7
- 230000000694 effects Effects 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 75
- 239000007788 liquid Substances 0.000 claims description 55
- 239000000706 filtrate Substances 0.000 claims description 44
- 238000003756 stirring Methods 0.000 claims description 44
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 38
- 229960002897 heparin Drugs 0.000 claims description 35
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 33
- 239000012530 fluid Substances 0.000 claims description 33
- 239000000463 material Substances 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 238000001556 precipitation Methods 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 239000008213 purified water Substances 0.000 claims description 20
- 230000001105 regulatory effect Effects 0.000 claims description 20
- 230000003647 oxidation Effects 0.000 claims description 18
- 238000007254 oxidation reaction Methods 0.000 claims description 18
- 239000002244 precipitate Substances 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- 239000012535 impurity Substances 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 17
- 238000001914 filtration Methods 0.000 claims description 16
- 230000008569 process Effects 0.000 claims description 13
- YMHOBZXQZVXHBM-UHFFFAOYSA-N 2,5-dimethoxy-4-bromophenethylamine Chemical compound COC1=CC(CCN)=C(OC)C=C1Br YMHOBZXQZVXHBM-UHFFFAOYSA-N 0.000 claims description 10
- 241000545067 Venus Species 0.000 claims description 10
- 239000013078 crystal Substances 0.000 claims description 10
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 9
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 9
- 239000001632 sodium acetate Substances 0.000 claims description 9
- 235000017281 sodium acetate Nutrition 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 8
- 230000001276 controlling effect Effects 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 230000001590 oxidative effect Effects 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 5
- -1 millipore filtration Chemical compound 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 229940076286 cupric acetate Drugs 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims 2
- 238000007710 freezing Methods 0.000 abstract description 5
- 230000002785 anti-thrombosis Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 230000001858 anti-Xa Effects 0.000 abstract description 2
- 239000003054 catalyst Substances 0.000 abstract description 2
- 108090000526 Papain Proteins 0.000 abstract 1
- 239000004365 Protease Substances 0.000 abstract 1
- 108010032819 exoribonuclease II Proteins 0.000 abstract 1
- 229940055729 papain Drugs 0.000 abstract 1
- 235000019834 papain Nutrition 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 36
- 239000002253 acid Substances 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
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- 238000004519 manufacturing process Methods 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- 239000003055 low molecular weight heparin Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 125000004103 aminoalkyl group Chemical group 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 229960004762 parnaparin Drugs 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
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- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 206010062713 Haemorrhagic diathesis Diseases 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a method for preparing high-quality low molecular parnaparin sodium, belonging to the field of biological medicine. The method comprises the following steps: using a coarse-product heparin sodium as a raw material, carrying out enzymolysis to the coarse-product heparin sodium through papain and ribonuclease II, oxidation-decoloring and purifying to obtain competitive-product heparin sodium; after dissolving the competitive-product heparin sodium, conducing degradation reaction under the action of a copper acetate catalyst and hydrogen peroxide; after completing the degradation reaction, using a heavy metal absorbent to remove Cu2+ or removing Cu2+ in an ultra-filtration mode, and then separating though a low molecular membrane to obtain a parnaparin sodium coarse-product with specific molecular weight; after decoloring and, alcohol-precipitating the parnaparin sodium coarse-product, and vacuum-freezing and drying to obtain a low molecular parnaparin sodium finished-product with the anti-Xa factor activity being 80-i10 IU/mg, the pH value being 5.5-8.0 and the average molecular weight being 4000-6000D.The preparation process is simple, the stability and antithrombotic activity of the product are good.
Description
Technical field
The present invention relates to biomedicine field, specifically the preparation method of a kind of high-quality low that heparin of molecule handkerchief (Parnaparin).
Background technology
Heparin is the Sulfated glycosaminoglycan of a kind of height, with external anticoagulation and anti thrombotic action is arranged in vivo.But the defects such as unfractionated heparin exists, and bioavailability is low, side effect is large and time transformation period is short, that heparin of handkerchief is a kind of of its regeneration product Low molecular heparin, there is Half-life in vivo prolongation, bioavailability advantages of higher, have lower hemorrhage risk, be the antithrombotic anticoagulation medicine of current main flow simultaneously.
Low that heparin of molecule handkerchief is after heparin is dissolved into certain concentration, adds certain sodium-chlor and sodium acetate, under specific temperature and pH condition, adds the cupric acetate catalyzed agent, by hydrogen peroxide degradation, forms.It is compared with the heparin product, and better antithrombotic acitivity is arranged, less hemorrhage risk, and lower on hematoblastic impact.
" a kind of production method of parnaparin " disclosed in the patent documentation that publication number is CN101942039A.The method by adding Cu in refined heparin sodium solution
2+Catalyzer, under specified temp and acidic conditions, adopt the hydrogen peroxide edman degradation Edman to obtain Low molecular heparin.In its preparation process, be to take refined heparin sodium as raw material, be difficult to control from the raw material source final finished quality; Due at Cu
2+Existence under, heparin reacts under alkaline condition with superoxide and degrades.This DeR has larger randomness, that glycosidic link between uronic acid or glycosamine all may rupture, thereby the Low molecular heparin sample that causes preparing have purity not, molecular weight distribution do not concentrate, the residual shortcoming such as too much of impurity, and then can affect the stability of preparation medicine, in use easily produce certain risk.And after degraded to metal Cu
2+In the removal process, use be the 001x7 resin, can cause product also partly to be adsorbed, reduced the yield of product.These are all the problems that current technique cann't be solved.
Summary of the invention
Technical assignment of the present invention is for above-mentioned the deficiencies in the prior art, and a kind of preparation method of high-quality low that heparin of molecule handkerchief is provided.
Technical assignment of the present invention is realized in the following manner: a kind of preparation method of high-quality low that heparin of molecule handkerchief is characterized in that:
Take crude heparin sodium as raw material, and crude heparin sodium is through papoid, rnase II enzymolysis, and oxidative decoloration, purification obtain refined heparin sodium;
Refined heparin sodium is carried out DeR after dissolving under cupric acetate catalyzed agent, hydrogen peroxide effect; After DeR completes, with the method for heavy metal absorbent or ultrafiltration, remove Cu
2+, and obtain that heparin crude product of handkerchief with specified molecular weight by membrane sepn;
That heparin crude product of handkerchief obtains low that heparin finished product of molecule handkerchief after decolouring, alcohol precipitation, vacuum lyophilization.
Aforesaid method comprises the following steps:
A) enzymolysis: to the water dissolution that adds 10～12 times of amounts in crude heparin sodium, regulate material liquid pH to 8.0～8.5, controlling feed temperature is 35 ℃～40 ℃, add appropriate papoid and rnase II, enzyme digestion reaction 8～10 hours, the centrifugal precipitation of going, supernatant liquid filtering, collect filtrate;
B) oxidative decoloration: control step a) middle filtrate temperature is 30 ℃～40 ℃, and adjusting filtrate pH is 9～11, adds the hydrogen peroxide of filtrate volume 0.3%～0.5% (v/v), and stirring reaction 4-6 hour under temperature control control pH, obtain refined solution; Regulating refined solution pH is 6～8, adds appropriate sodium-chlor, millipore filtration, and alcohol precipitation, separate to obtain throw out; The mass volume ratio of described sodium-chlor and refined solution is 0.8%～1% (w/v);
C) in the throw out obtained ultrafiltration removal of impurities: to step b), add the purified water of 20～25 times of amounts to dissolve, the ultra-filtration membrane that the molecular weight cut-off of take after dissolving fully is 3000～10000Da carries out 5～8 hours tangential flow loop ultrafiltrations, collects trapped fluid;
D) alcohol precipitation removal of impurities: the removal of impurities of trapped fluid alcohol precipitation, the gained drying precipitate is weighed;
E) degraded: take purified water dissolving step d), the throw out of gained obtains the heparin sodium aqua of concentration as 3%～5% (w/v), add appropriate sodium-chlor and sodium acetate in solution, regulate pH to 7.5～8.5, add again the hydrogen peroxide of the appropriate venus crystals aqueous solution and material liquid volume 2.5%～3.5% (v/v), carry out DeR under 50～55 ℃;
The consumption of described sodium-chlor is steps d) 0.5%～1% (w/w) of gained weight of precipitate;
The consumption of described sodium acetate is steps d) 1.5%～2% (w/w) of gained weight of precipitate;
The consumption of described venus crystals is steps d) 2.6%～2.8% (w/w) of gained weight of precipitate;
The concentration of the venus crystals aqueous solution is 4% (w/v);
F) removal heavy metal: utilize heavy metal absorbent or ultra-filtration technique to remove the heavy metal in feed liquid;
G) ultrafiltration: regulate material liquid pH to 6.8～7.2 after removal heavy metal, the ultra-filtration membrane loop ultrafiltration that is 1000Da～3000Da with molecular weight cut-off is controlled molecular weight, until molecular weight reaches control criterion, collects trapped fluid; The trapped fluid alcohol precipitation, separate to obtain throw out;
H) oxidative decoloration: to step g) in the gained throw out, add the water stirring of 8～10 times of amounts to make its dissolving, regulating material liquid pH is 9～10, the hydrogen peroxide that adds material liquid volume 1.2%～1.5% (v/v), under 20～30 ℃ of feed temperatures, stirring reaction is 6～8 hours; After oxidation, regulating material liquid pH is 6～8, adds wherein appropriate sodium-chlor, millipore filtration, and alcohol precipitation, separate to obtain throw out; The mass volume ratio of described sodium-chlor and feed liquid is 1.0%～1.2% (w/v);
I) freeze-drying: add 10～12 times of amount purified water to be dissolved in throw out, dissolved the degerming membrane filtration through 0.1 μ m, collect filtrate, and regulate filtrate pH value to 6.4～7.4; Filtrate is carried out to frozen dried, De Pana heparin finished product.
" until molecular weight reaches control criterion " of mentioning in step g: molecular-weight average is: 4000-6000, and molecular weight is less than 3000 ratio and is :≤30%, the ratio that molecular weight is 3000-8000 is: 50%～60%.
The preferable amount of described papoid and rnase II is 4%～6% of crude heparin sodium weight.
Step b) in, the detailed process of alcohol precipitation is: after millipore filtration completes, and to 95% ethanol that adds 1.0～1.2 times of volumes in filtrate, the last stirring standing 10～12 hours.
Steps d) detailed process of alcohol precipitation removal of impurities is: to step c) add appropriate sodium-chlor in the gained trapped fluid, then add 95% ethanol of 1～1.2 times of volume, and precipitate 10～12 hours, separate to obtain throw out, the gained drying precipitate is weighed; The mass volume ratio of described sodium-chlor and feed liquid is 0.8%～1% (w/v).
Preferred following methods removal step e) heavy metal in the gained feed liquid:
Method one: step e) DeR is regulated the pH value to neutral (7.5～8.5) after completing, and adds appropriate heavy metal absorbent, and regulating feed temperature is 30 ℃～35 ℃, stirring reaction 1.5～2 hours, react rear millipore filtration, collected filtrate, completed the removal heavy metal operation; The consumption of described heavy metal absorbent is steps d) 4%～6% (w/w) of gained weight of precipitate.
Method two: step e) DeR is regulated the pH value to neutral (7.5～8.5) after completing, and the ultra-filtration membrane that is 600～1000Da with molecular weight cut-off carries out loop ultrafiltration, collects trapped fluid, completes the removal heavy metal operation.
Step g) in, the detailed process of alcohol precipitation is: to 95% ethanol that adds 5～7 times of volumes in trapped fluid, and after stirring 20～30 minutes, standing 8～10 hours.
Step h) in, the detailed process of alcohol precipitation is: after millipore filtration completes, 95% ethanol to adding 4～6 times of volumes in filtrate, after stirring 20～30 minutes, staticly settle 6～8 hours.
Compared with prior art, the preparation method of high-quality low that heparin of molecule handkerchief of the present invention has following outstanding beneficial effect:
(1) take crude heparin sodium as raw material, avoided preparing and falling behind loaded down with trivial details treating processes from the crude product to the elaboration in traditional technology, and increased the controllability of raw material sources;
(2) metal catalyst simple to operate, as to add while utilizing heavy metal absorbent or advanced ultra-filtration technique to process degraded, and obtained the product with stable molecule amount by ultrafiltration, whole technique advanced person is feasible, can realize suitability for industrialized production.
(3) molecular-weight average of low that heparin of molecule handkerchief prepared by the inventive method is 4000～6000 dalton, and it is≤30.0% that molecular weight is less than 3000 ratio; Molecular weight is 50.0%～60.0% in 3000～8000 ratio; Anti-Xa is that 80IU/mg-110IU/mg, pH value are 6.4～7.4, nitrogen content (dry product): 1.5%～2.5%, SO4 because of activity
2-/ COO-ratio:>=1.9, ethanol content≤3000ppm, quality surmounts the European Pharmacopoeia standard, and quality stability is good, and bioavailability is high, and the Half-life in vivo time is long, has very large promotion prospect.
The accompanying drawing explanation
Accompanying drawing 1 is the process flow sheet of the embodiment of the present invention one, two;
Accompanying drawing 2 is process flow sheets of the embodiment of the present invention three, four.
Embodiment
In conjunction with Figure of description, with specific embodiment, the preparation method to high-quality low that heparin of molecule handkerchief of the present invention is described in detail below.
Preparation Example one:
A) enzymolysis: get crude heparin sodium 800g, add wherein the 8kg water dissolution, regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 8.4, and controlling feed temperature is 37 ℃.Add 42g papoid, 36g rnase II in feed liquid, stirring reaction 8 hours, the centrifugal precipitation of going, filter supernatant liquor with plate filter, collect filtrate 7.6L;
B) oxidation: control step a) middle filtrate temperature is 32 ℃, and adjusting material liquid pH is 9.7, adds the 35ml hydrogen peroxide, and under temperature control control pH, stirring reaction is 5 hours, obtains refined solution; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 6.9, after adding wherein 72g sodium-chlor to dissolve, filtered with the millipore filter of 0.65um, add again after filtration in filtrate and add 8L95% ethanol, stir latter standing 10 hours.Standing completing, pump supernatant, separates to obtain throw out;
C) ultrafiltration removal of impurities: will in the throw out obtained in step b, add the 16kg purified water to be dissolved, after dissolving fully, carry out cross-flow ultrafiltration, the ultra-filtration membrane that the selective retention molecular weight is 10000Da carries out loop ultrafiltration 6 hours, collects trapped fluid 9L;
D) alcohol precipitation removal of impurities: add 78g sodium-chlor in trapped fluid, then add 10L95% ethanol, precipitate 10 hours, separate to obtain throw out, by throw out vacuum-drying and weigh, the heavy 716g of throw out;
E) degraded: to steps d), in the throw out of gained, add the 21kg purified water to dissolve, add 4g sodium-chlor and 11g sodium acetate in feed liquid, regulate the pH8.1 of feed liquid with 20% (w/v) sodium hydroxide solution, the venus crystals aqueous solution that the concentration that adds 460ml to dissolve is 4% (w/v) and the hydrogen peroxide of 600ml carry out DeR 10h under 54 ℃.
F) duplicate removal metal: having degraded with the salt acid for adjusting pH value of 4mol/L is 6.9, adds the 30g heavy metal absorbent, and regulating feed temperature is 30 ℃, and stirring reaction 2h, reacted the rear filter membrane with 0.65um and filtered.Heavy metal absorbent is the Aminoalkyl 2 Functionalised Silica heavy metal absorbents that EASTAR chemical corp company produces.
G) ultrafiltration: filtered with the film loop ultrafiltration of 1000Da and controlled molecular weight, until molecular weight reaches the standard of control, (molecular-weight average is: 4000-6000, molecular weight is less than 3000 ratio :≤30%, the ratio that molecular weight is 3000-8000 is: 50%～60%), collect trapped fluid; Add wherein 140L95% ethanol, after stirring 20 minutes, standing 9 hours;
H) throw out oxidation: by step g) removes supernatant, adds 5.9kg water to stir and makes its dissolving, and regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 9.3, adds the 70ml hydrogen peroxide, and under 25 ℃ of feed temperatures, stirring reaction is 6 hours; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 6.6, add wherein 58g sodium-chlor, filtered with the millipore filter of 0.22um, filtered and added wherein 25L95% ethanol, after stirring 25 minutes, after staticly settling 7 hours, remove supernatant; Trapped fluid, after the degerming membrane filtration of 0.1 μ m, is obtained to filtrate, then regulate filtrate pH to 6.9 with 1mol/L HCl solution;
I) freeze-drying: to h) add the 14kg purified water to be dissolved, after dissolving fully, filtrate is transferred in vacuum freezing drying oven and carries out frozen dried, De Pana heparin finished product 603g.
Preparation Example two:
A) enzymolysis: get crude heparin sodium 1000g, add wherein the 12kg water dissolution, regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 8.2, and controlling feed temperature is 36 ℃.Add 60g papoid, 48g rnase II in feed liquid, stirring reaction 9 hours, the centrifugal precipitation of going, filter supernatant liquor with plate filter, collect filtrate 972L;
B) oxidation: control step a) middle filtrate temperature is 36 ℃, and adjusting material liquid pH is 10.2, adds the 46ml hydrogen peroxide, and under temperature control control pH, stirring reaction is 5 hours, obtains refined solution; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 7.1, after adding wherein 90g sodium-chlor to dissolve, filtered with the millipore filter of 0.65um, add again after filtration in filtrate and add 11L95% ethanol, stir latter standing 10 hours.Standing completing, pump supernatant, separates to obtain throw out;
C) ultrafiltration removal of impurities: will in the throw out obtained in step b, add the 20kg purified water to be dissolved, after dissolving fully, carry out cross-flow ultrafiltration, the ultra-filtration membrane that the selective retention molecular weight is 10000Da carries out loop ultrafiltration 7 hours, collects trapped fluid 11L;
D) alcohol precipitation removal of impurities: add 100g sodium-chlor in trapped fluid, then add 11L95% ethanol, precipitate 11 hours, separate to obtain throw out, by throw out vacuum-drying and weigh, the heavy 907g of throw out;
E) degraded: to steps d), in the throw out of gained, add the 23kg purified water to dissolve, add 7.2g sodium-chlor and 18g sodium acetate in feed liquid, regulate the pH7.9 of feed liquid with 20% (w/v) sodium hydroxide solution, the venus crystals aqueous solution that the concentration that adds 620ml to dissolve is 4% (w/v) and the hydrogen peroxide of 690ml carry out DeR 11h under 54 ℃.
F) duplicate removal metal: having degraded with the salt acid for adjusting pH value of 4mol/L is 7.0, adds the 48g heavy metal absorbent, and regulating feed temperature is 32 ℃, and stirring reaction 2h, reacted the rear filter membrane with 0.65um and filtered; Heavy metal absorbent is the Aminoalkyl 2 Functionalised Silica heavy metal absorbents that EASTAR chemical corp company produces.
G) ultrafiltration: filtered with the film loop ultrafiltration of 1000Da and controlled molecular weight, until molecular weight reaches the standard of control, (molecular-weight average is: 4000-6000, molecular weight is less than 3000 ratio :≤30%, the ratio that molecular weight is 3000-8000 is: 50%～60%), collect trapped fluid; Add wherein 145L95% ethanol, after stirring 25 minutes, standing 9 hours;
H) throw out oxidation: by step g) removes supernatant, adds 8.0kg water to stir and makes its dissolving, and regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 9.5, adds the 115ml hydrogen peroxide, and under 30 ℃ of feed temperatures, stirring reaction is 7 hours; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 7.1, add wherein 60g sodium-chlor, filtered with the millipore filter of 0.22um, filtered and added wherein 43L95% ethanol, after stirring 20 minutes, after staticly settling 7.5 hours, remove supernatant.
I) freeze-drying: to h), add the 17kg purified water to be dissolved; After dissolving the degerming membrane filtration by 0.1 μ m fully, obtain filtrate, then regulate filtrate pH to 6.7 with 1mol/L HCl solution; Filtrate is transferred in vacuum freezing drying oven and carries out frozen dried, De Pana heparin finished product 778g.
Preparation Example three:
A) enzymolysis: get crude heparin sodium 800g, add wherein the 8kg water dissolution, regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 8.1, and controlling feed temperature is 36 ℃.Add 45g papoid, 36g rnase II in feed liquid, stirring reaction 8 hours, the centrifugal precipitation of going, filter supernatant liquor with plate filter, collect filtrate 7.8L;
B) oxidation: control step a) middle filtrate temperature is 34 ℃, and adjusting material liquid pH is between 9.8, adds the 26ml hydrogen peroxide, and under temperature control control pH, stirring reaction is 4.5 hours, obtains refined solution; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 6.8, after adding wherein 63g sodium-chlor to dissolve, filtered with the millipore filter of 0.65um, add again after filtration in filtrate and add 8L95% ethanol, stir latter standing 10 hours.Standing completing, pump supernatant, separates to obtain throw out;
C) ultrafiltration removal of impurities: will in the throw out obtained in step b, add the 16kg purified water to be dissolved, after dissolving fully, carry out cross-flow ultrafiltration, the ultra-filtration membrane that the selective retention molecular weight is 10000Da carries out loop ultrafiltration 6 hours, collects trapped fluid 9L;
D) alcohol precipitation removal of impurities: add 72g sodium-chlor in trapped fluid, then add 9L95% ethanol, precipitate 10 hours, separate to obtain throw out, by throw out vacuum-drying and weigh, the heavy 720g of throw out;
E) degraded: to steps d), in the throw out of gained, add the 24kg purified water to dissolve, add 4g sodium-chlor and 11g sodium acetate in feed liquid, regulate the pH7.8 of feed liquid with 20% (w/v) sodium hydroxide solution, the venus crystals aqueous solution that the concentration that adds 475ml to dissolve is 4% (w/v) and the hydrogen peroxide of 600ml carry out DeR 10h under 52 ℃.
F) removal heavy metal: having degraded with the salt acid for adjusting pH value of 4mol/L is 6.9, with the ultra-filtration membrane of 600Da, carries out loop ultrafiltration, collects ultrafiltration trapped fluid 18L.
G) ultrafiltration: add purified water 6L in trapped fluid, with the ultra-filtration membrane of 1000Da, carry out loop ultrafiltration control molecular weight, until molecular weight reaches the standard of control, collect trapped fluid, in trapped fluid, add 120L95% ethanol, after stirring 20 minutes, standing 8 hours;
H) throw out oxidation: by step g) removes supernatant, adds 5.8kg water to stir and makes its dissolving, and regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 9.0, adds the 70ml hydrogen peroxide, and under 25 ℃ of feed temperatures, stirring reaction is 6 hours; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 6.6, add wherein 60g sodium-chlor, filtered with the millipore filter of 0.22um, filtered and added wherein 28L95% ethanol, after stirring 30 minutes, after staticly settling 7 hours, remove supernatant.I) freeze-drying: to step h), add the 14kg purified water to be dissolved, after dissolving fully, with the degerming membrane filtration of 0.1 μ m, obtain filtrate, then be 6.8 by 1mol/L HCl solution adjusting filtrate pH value; Filtrate is transferred in vacuum freezing drying oven and carries out frozen dried, De Pana heparin finished product 610g.
Preparation Example four:
A) enzymolysis: get crude heparin sodium 1000g, add wherein the 12kg water dissolution, regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 8.3, and controlling feed temperature is 37 ℃.Add 50g papoid, 40g rnase II in feed liquid, stirring reaction 9 hours, the centrifugal precipitation of going, filter supernatant liquor with plate filter, collect filtrate 970L;
B) oxidation: control step a) middle filtrate temperature is 36 ℃, and adjusting material liquid pH is between 10.4, adds the 35ml hydrogen peroxide, and under temperature control control pH, stirring reaction is 5.5 hours, obtains refined solution; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 7.2, after adding wherein 90g sodium-chlor to dissolve, filtered with the millipore filter of 0.65um, add again after filtration in filtrate and add 11L95% ethanol, stir latter standing 11 hours.Standing completing, pump supernatant, separates to obtain throw out;
C) ultrafiltration removal of impurities: will in the throw out obtained in step b, add the 20kg purified water to be dissolved, after dissolving fully, carry out cross-flow ultrafiltration, the ultra-filtration membrane that the selective retention molecular weight is 10000Da carries out loop ultrafiltration 7 hours, collects trapped fluid 11L;
D) alcohol precipitation removal of impurities: add 100g sodium-chlor in trapped fluid, then add 12L95% ethanol, precipitate 11 hours, separate to obtain throw out, by throw out vacuum-drying and weigh, the heavy 910g of throw out;
E) degraded: to steps d), in the throw out of gained, add the 28kg purified water to dissolve, add 7.3g sodium-chlor and 18g sodium acetate in feed liquid, regulate the pH7.8 of feed liquid with 20% (w/v) sodium hydroxide solution, the venus crystals aqueous solution that the concentration that adds 615ml to dissolve is 4% (w/v) and the hydrogen peroxide of 690ml carry out DeR 11h under 54 ℃.
F) removal heavy metal: having degraded with the salt acid for adjusting pH value of 4mol/L is 7.1, with the ultra-filtration membrane of 600Da, carries out loop ultrafiltration, collects ultrafiltration trapped fluid 19L.
G) ultrafiltration: add purified water 7L in trapped fluid, with the ultra-filtration membrane of 1000Da, carry out loop ultrafiltration control molecular weight, until molecular weight reaches the standard of control, collect trapped fluid, in trapped fluid, add 140L95% ethanol, after stirring 25 minutes, standing 9 hours;
H) throw out oxidation: by step g) removes supernatant, adds 8.1kg water to stir and makes its dissolving, and regulating material liquid pH with 20% (w/v) sodium hydroxide solution is 9.3, adds the 113ml hydrogen peroxide, and under 30 ℃ of feed temperatures, stirring reaction is 7 hours; After oxidation, by the HCl adjusting material liquid pH of 4mol/L, be 7.3, add wherein 60g sodium-chlor, filtered with the millipore filter of 0.22um, filtered and added wherein 45L95% ethanol, after stirring 20 minutes, after staticly settling 7 hours, remove supernatant.
I) freeze-drying: to step h), add the 15kg purified water to be dissolved, after dissolving fully, with the degerming membrane filtration of 0.1 μ m, obtain filtrate, then be 6.9 by 1mol/L HCl solution adjusting filtrate pH value; Filtrate is transferred in vacuum freezing drying oven and carries out frozen dried, De Pana heparin finished product 782g.
Test example: with reference to the American Pharmacopeia method, sample is carried out to activity and detect
High-quality low that heparin of molecule handkerchief of Preparation Example one, two, three, four gained of take is tested as sample, and test-results sees the following form:
From above-mentioned detected result, no matter that heparin of handkerchief that present method is produced is in product appearance in shape, or in interior domestic current patent and the import standard thereof of all being better than on quality product.The product of producing has the characteristics such as bioavailability is high, Half-life in vivo is long, bleeding tendency is little.Its simple in production process operation, be easy to test suitability for industrialized production.
Claims (9)
1. the preparation method of high-quality low that heparin of molecule handkerchief is characterized in that:
Take crude heparin sodium as raw material, and crude heparin sodium is through papoid, rnase II enzymolysis, and oxidative decoloration, purification obtain refined heparin sodium;
Refined heparin sodium is carried out DeR after dissolving under cupric acetate catalyzed agent, hydrogen peroxide effect; After DeR completes, with heavy metal absorbent or ultra-filtration technique, remove Cu
2+, and control molecular weight by membrane sepn, De Pana heparin crude product after alcohol precipitation;
That heparin crude product of handkerchief obtains low that heparin finished product of molecule handkerchief after decolouring, alcohol precipitation, vacuum lyophilization.
2. preparation method according to claim 1 is characterized in that comprising the following steps:
A) enzymolysis: to the water dissolution that adds 10～12 times of amounts in crude heparin sodium, regulate material liquid pH to 8.0～8.5, controlling feed temperature is 35 ℃～40 ℃, add appropriate papoid and rnase II, enzyme digestion reaction 8～10 hours, the centrifugal precipitation of going, supernatant liquid filtering, collect filtrate;
B) oxidative decoloration: control step a) middle filtrate temperature is 30 ℃～40 ℃, and adjusting filtrate pH is 9～11, adds the hydrogen peroxide of filtrate volume 0.3%～0.5%, and under temperature control control pH, stirring reaction is 4～6 hours, obtains refined solution; Regulating refined solution pH is 6～8, adds appropriate sodium-chlor, millipore filtration, and alcohol precipitation, separate to obtain throw out; The mass volume ratio of described sodium-chlor and refined solution is 0.8%～1%;
C) in the throw out obtained ultrafiltration removal of impurities: to step b), add the purified water of 20～25 times of amounts to dissolve, the ultra-filtration membrane that the molecular weight cut-off of take after dissolving fully is 3000～10000Da carries out 5～8 hours tangential flow loop ultrafiltrations, collects trapped fluid;
D) alcohol precipitation removal of impurities: the removal of impurities of trapped fluid alcohol precipitation, the gained drying precipitate is weighed;
E) degraded: take purified water dissolving step d), the throw out of gained obtains the heparin sodium aqua that concentration is 3%～5%, add appropriate sodium-chlor and sodium acetate in solution, regulate pH to 7.5～8.5, the hydrogen peroxide that adds again the appropriate venus crystals aqueous solution and material liquid volume 2.5%～3.5% carries out DeR under 50～55 ℃;
The consumption of described sodium-chlor is steps d) the gained weight of precipitate 0.5%～1%;
The consumption of described sodium acetate is steps d) the gained weight of precipitate 1.5%～2%;
The consumption of described venus crystals is steps d) the gained weight of precipitate 2.6%～2.8%;
The concentration of the venus crystals aqueous solution is 4%;
F) removal heavy metal: utilize heavy metal absorbent or ultra-filtration technique to remove the heavy metal in feed liquid;
G) ultrafiltration: regulate material liquid pH to 6.8～7.2 after removal heavy metal, the ultra-filtration membrane loop ultrafiltration that is 1000Da～3000Da with molecular weight cut-off is controlled molecular weight, until molecular weight reaches control criterion, collects trapped fluid; The trapped fluid alcohol precipitation, separate to obtain throw out;
H) oxidative decoloration: to step g) in the gained throw out, add the water stirring of 8～10 times of amounts to make its dissolving, regulating material liquid pH is 9～10, adds the hydrogen peroxide of material liquid volume 1.2%～1.5%, and under 20～30 ℃ of feed temperatures, stirring reaction is 6～8 hours; After oxidation, regulating material liquid pH is 6～8, adds wherein appropriate sodium-chlor, millipore filtration, and alcohol precipitation, separate to obtain throw out; The mass volume ratio of described sodium-chlor and feed liquid is 1.0%～1.2%;
I) freeze-drying: add 10～12 times of amount purified water to be dissolved in throw out, dissolved the degerming membrane filtration through 0.1 μ m, collect filtrate, and regulate filtrate pH value to 6.4～7.4; Filtrate is carried out to frozen dried, De Pana heparin finished product.
3. the preparation method of high-quality low that heparin of molecule handkerchief according to claim 1 and 2, is characterized in that, the consumption of described papoid and rnase II is 3%～5% of crude heparin sodium weight.
4. the preparation method of high-quality low that heparin of molecule handkerchief according to claim 2, it is characterized in that, step b) in, the detailed process of alcohol precipitation is: after millipore filtration completes, and to 95% ethanol that adds 1.0～1.2 times of volumes in filtrate, the last stirring standing 10～12 hours.
5. the preparation method of high-quality low that heparin of molecule handkerchief according to claim 2, it is characterized in that, steps d) detailed process of alcohol precipitation removal of impurities is: to step c) add appropriate sodium-chlor in the gained trapped fluid, 95% ethanol that adds again 1～1.2 times of volume, precipitate 10～12 hours, separate to obtain throw out, the gained drying precipitate is weighed; The mass volume ratio of described sodium-chlor and feed liquid is 0.8%～1%;
6. the preparation method of high-quality low that heparin of molecule handkerchief according to claim 2, it is characterized in that, step f) utilize heavy metal absorbent to remove the heavy metal in feed liquid: to regulate the pH value after step e DeR completes to neutral, add appropriate heavy metal absorbent, regulating feed temperature is 30 ℃～35 ℃, stirring reaction 1.5～2 hours, reacted rear millipore filtration, collects filtrate;
The consumption of described heavy metal absorbent is steps d) the gained weight of precipitate 4%～6%.
7. the preparation method of high-quality low that heparin of molecule handkerchief according to claim 2, it is characterized in that, step f) utilize ultra-filtration technique to remove the heavy metal in feed liquid: to regulate the pH value after step e DeR completes to neutral, the ultra-filtration membrane that is 600～1000Da with molecular weight cut-off carries out loop ultrafiltration, collects trapped fluid.
8. the preparation method of high-quality low that heparin of molecule handkerchief according to claim 2, is characterized in that step g) in the detailed process of alcohol precipitation be: to 95% ethanol that adds 5～7 times of volumes in trapped fluid, after stirring 20～30 minutes, standing 8～10 hours.
9. the preparation method of high-quality low that heparin of molecule handkerchief according to claim 2, it is characterized in that step h) in the detailed process of alcohol precipitation be: after millipore filtration completes, to 95% ethanol that adds 4～6 times of volumes in filtrate, stir after 20～30 minutes, staticly settle 6～8 hours.
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Denomination of invention: A preparation method of high quality low molecular weight panaxaparin Effective date of registration: 20220630 Granted publication date: 20150812 Pledgee: Industrial and Commercial Bank of China Limited Jining urban sub branch Pledgor: SHANDONG CHENLONG PHARMACEUTICAL Co.,Ltd. Registration number: Y2022980009589 |