CN109776696A - A kind of preparation process of high purity heparin sodium - Google Patents
A kind of preparation process of high purity heparin sodium Download PDFInfo
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- CN109776696A CN109776696A CN201910035877.7A CN201910035877A CN109776696A CN 109776696 A CN109776696 A CN 109776696A CN 201910035877 A CN201910035877 A CN 201910035877A CN 109776696 A CN109776696 A CN 109776696A
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Abstract
The invention discloses a kind of preparation processes of high purity heparin sodium, specifically includes the following steps: S1, enzymatic hydrolysis salt solution removing protein: the purified water of 10-12 times of heparin sodium crude weight is warming up to 50.0-60.0 DEG C, S2, once oxidation, S3, precipitating, S4, secondary oxidation, S5, it precipitates again, S6, freeze-drying, the present invention relates to pharmaceutical technology fields.The preparation process of the high purity heparin sodium, it can be achieved by way of taking enzymatic hydrolysis salt solution integral type dissociation, in high temperature dissociation process, heparin is protected not to be damaged, different impurities are removed with different concentration ethanol, in oxidation process, natrium carbonicum calcinatum and anhydrous sodium sulfate is added, it is prepared with special ratios, it solves the problems, such as to be difficult to obtain the heparin sodium of high-purity because generating destruction to heparin sodium structure when the purification of existing heparin sodium, HPLC method is used simultaneously, the impurity removed in each step is detected, it is easy to operate, product yield is high, more effectively control final product quality.
Description
Technical field
The present invention relates to pharmaceutical technology field, specially a kind of preparation process of high purity heparin sodium.
Background technique
Heparin is widely present in lactation and moves in dynamic internal organ and intestinal mucosa, mainly by d-glucosamine, L- iduronic acid, M-
The mucopolysaccharide sulfuric ester that acetylglucosamine and D- glucuronic acid alternately form, as a kind of natural anticoagulative substance, anticoagulant
Blood promotes matter albumen enzyme r e lease and the molten cell system of complement etc. to have good activity, is widely used in epidemic encephalitis, loses
The treatment such as mass formed by blood stasis, blood embolization, acute myocardial infarction, artery sclerosis, heparin is soluble easily in water, organic molten insoluble in ethyl alcohol, acetone etc.
Agent contains 5 sulfates and 2 hydroxyls in molecular structure unit, is in highly acid, is polyanion, can occur with cation anti-
Salt should be generated, since heparin mainly exists in the form of heparin sodium-protein complex in animal body, so that current animal
A certain amount of protein is remained in the crude heparin sodium extracted in body, and certain influence is generated on the activity of heparin sodium.
The subtractive process of heparin mainly carries out the purification of the substances such as removing protein, nucleic acid and decoloration to crude heparin sodium at present,
Reach National Pharmacopeia standard requirements, has delivered many methods except serine in heparin both at home and abroad, but be both needed to separate specific point
The heparin fragment of son amount, then removes the serine residue of specific heparin fragment, is unable to reach industrialization production requirements, and existing
Heparin method for extraction and purification takes the substances such as soda acid processing removal impurity protein, nucleic acid mostly, using potassium permanganate oxidation and mistake
Hydrogen oxide oxidation removal colors, but potassium permanganate oxidation method can lead to the problem of manganese dioxide absorption heparin, make liver
Plain loss of activity is big, leads to that the heparin sodium rate of recovery is low, shade deviation, and hydrogen peroxide oxidation process is wide at present because product color is preferable
General application, but since the prior art is difficult to control oxidant additional amount and reaction condition, it can often be added in actual production
Excessive oxidant, and reaction condition is also not suitable for, and finally to heparin sodium structural damage, keeps its yield low, activity is low, it is difficult to
Obtain the refined heparin sodium product of higher degree.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the present invention provides a kind of preparation processes of high purity heparin sodium, solve existing
It is difficult to obtain the heparin sodium of high-purity because generating destruction to heparin sodium structure when heparin sodium refines, easily heparin sodium structure is caused
It destroys, keeps its yield low, the low problem of activity.
(2) technical solution
In order to achieve the above object, the present invention is achieved by the following technical programs: a kind of preparation of high purity heparin sodium
Technique, specifically includes the following steps:
S1, enzymatic hydrolysis salt solution removing protein: being warming up to 50.0-60.0 DEG C for the purified water of 10-12 times of heparin sodium crude weight,
The sausage-casing salt of purifying water volume 2.0%-5.0% is added under stirring, heparin sodium crude is added after dissolution, continues stirring and dissolving
At least 1 hour, after dissolution, feed liquid temperature is down to 35.0-45.0 DEG C, the pancreatin dissolved is added, stirs evenly, is controlled
35.0-45.0 DEG C of temperature processed, pH value is adjusted to 7-9 with sodium hydroxide solution, enzyme digestion reaction 3.0-5.0 hours is kept the temperature, material is added
Liquid 0.3% natrium carbonicum calcinatum of product and 0.25% sodium hydrogensulfite, are warming up to 50.0-70.0 DEG C, and the nothing dissolved is added
Water calcium chloride is warming up to 80.0-90.0 DEG C, insulation reaction 30-60 minutes, natrium carbonicum calcinatum is added, insulated and stirred reacts 30-60
It filters after minute, filtering removal impurity is tested and analyzed with HPLC method；
S2, once oxidation: filtrate temperature is adjusted to 25.0-40.0 DEG C, adjusts material liquid pH with 4mol/L sodium hydroxide solution
Value adds the natrium carbonicum calcinatum of material liquid volume 0.3% and 0.25% anhydrous sodium sulfate, by material liquid volume to 10-11
2.0% is added sodium chloride, and the hydrogen peroxide oxidation 12-18h of material liquid volume 2.0% is added；
S3, precipitating: after oxidation, filtering feed liquid, by material liquid volume: ethyl alcohol volume 1:1.0-1:1.2 addition has warmed up
To 40.0-50.0 DEG C of ethyl alcohol, feed liquid concentration of alcohol 40 ± 2% is controlled, temperature is 35.0-40.0 DEG C, and heat preservation staticly settles 4
± 1h, pumps supernatant, and impurity in supernatant is sunk again, is tested and analyzed with HPLC method to heavy impurity again；
S4, secondary oxidation: the purified water of 6.9-7.3 times of heparin sodium crude weight being added into oxidation precipitation tank II, adjusts
Feed liquid temperature adjusts material liquid pH value to 10-11 to 25.0-30.0 DEG C, with 4mol/L sodium hydroxide solution, adds material liquid volume
0.3% natrium carbonicum calcinatum and 0.25% anhydrous sodium sulfate, stirring and dissolving, then by material liquid volume 2.0% be added sodium chloride,
The hydrogen peroxide oxidation 12-18h of material liquid volume 2.0% is added；
S5, it precipitates again: after oxidation, filtering feed liquid, by material liquid volume: ethyl alcohol volume=1:1.1-1:1.3 is added
It has warmed up to 40.0-50.0 DEG C of ethyl alcohol, controls feed liquid concentration of alcohol 46 ± 2%, temperature is 35.0-40.0 DEG C, and heat preservation is stood
4 ± 1h is precipitated, supernatant is pumped, impurity in supernatant is sunk again, heavy impurity again is tested and analyzed with HPLC method；
S6, freeze-drying: the purified water measured with 3 times of heparin sodium crude dissolves sediment sufficiently, will with 4mol/L hydrochloric acid
PH value is transferred to 6.0 ± 0.5, filters, and high purity heparin sodium can be obtained in freeze-drying.
Preferably, the ratio of trypsase is 1.2-1.8 times of crude product weight and potency ratio in the step S1.
Preferably, anhydrous calcium chloride concentration is 2.0%-3.0% in the step S1.
Preferably, Carbon Dioxide na concn is 2.0%-3.0% in the step S1.
Preferably, the original content of hydrogen peroxide is 30% in the step S2 and step S4.
The preparation method of heparin sodium provided by the invention, fusion enzymatic hydrolysis oxidation are integrated, and egg is added in crude heparin sodium
White enzyme digests the albumen wherein contained, filters off removing protein, then is precipitated by once oxidation, removal dermatan sulfate and
Chondroitin sulfate precipitates removal Heparan sulfate by secondary oxidation, in oxidation process, protective agent-Carbon Dioxide is added
Sodium and anhydrous sodium sulfate repeatedly aoxidize, heparin structure are avoided to be destroyed, while being incorporated into all that impurity separates on heparin
Removal, finally to obtained heparin sodium aqua, freeze-drying obtains sterling, the method maintains heparin structure integrality, gained
Heparin sodium purity is high, impurity is few, and in this invention, we have studied in enzymatic hydrolysis and oxidation process, protective agent is to product quality
Influence, it is determined that protectant importance, case study are as follows:
Case study 1: being warming up to 50.0 DEG C for the purified water of 10 times of heparin sodium crude weight, and purifying is added under stirring
Heparin sodium crude is added in the sausage-casing salt of water volume 2.0% after dissolution, continue stirring and dissolving 1 hour, is cooled to 35.0 DEG C, is added
The pancreatin dissolved, stirs evenly, and controls 35.0 DEG C of temperature, and the sodium hydroxide solution of 4mol/L is added dropwise, and adjusts pH value to 7, protects
Warm enzyme digestion reaction 3.0 hours；50.0 DEG C are warming up to, the anhydrous calcium chloride dissolved is added, is warming up to 80.0 DEG C, insulation reaction
30 minutes, 2.0% natrium carbonicum calcinatum of liquor capacity is added, insulated and stirred filters after reacting 30 minutes, precipitates, and sediment dehydration is dry
It is dry, remember sample 1.
Case study 2: being warming up to 50.0 DEG C for the purified water of 10 times of heparin sodium crude weight, and purifying is added under stirring
Heparin sodium crude is added in the sausage-casing salt of water volume 2.0% after dissolution, continue stirring and dissolving 1 hour, is cooled to 35.0 DEG C, is added
The pancreatin dissolved, stirs evenly, and controls 35.0 DEG C of temperature, and the sodium hydroxide solution of 4mol/L is added dropwise, and adjusts pH value to 7, protects
Warm enzyme digestion reaction 3.0 hours is added the natrium carbonicum calcinatum and 0.3% sodium hydrogensulfite of 0.3% concentration of liquor capacity, is warming up to
50.0 DEG C, the anhydrous calcium chloride dissolved is added, is warming up to 80.0 DEG C, insulation reaction 30 minutes, 2.0% nothing of liquor capacity is added
Aqueous sodium carbonate, insulated and stirred filter after reacting 30 minutes, precipitate, and sediment dehydration and drying remembers sample 2.
Case study 3: being warming up to 50.0 DEG C for the purified water of 10 times of heparin sodium crude weight, and purifying is added under stirring
Heparin sodium crude is added in the sausage-casing salt of water volume 2.0% after dissolution, continue stirring and dissolving 1 hour, is cooled to 35.0 DEG C, is added
The pancreatin dissolved, stirs evenly, and controls 35.0 DEG C of temperature, and the sodium hydroxide solution of 4mol/L is added dropwise, and adjusts pH value to 7, protects
Warm enzyme digestion reaction 3.0 hours is added the natrium carbonicum calcinatum and 0.25% sodium hydrogensulfite of 0.3% concentration of liquor capacity, is warming up to
50.0 DEG C, the anhydrous calcium chloride dissolved is added, is warming up to 80.0 DEG C, insulation reaction 30 minutes, 2.0% nothing of liquor capacity is added
Aqueous sodium carbonate, insulated and stirred filter after reacting 30 minutes, precipitate, and sediment dehydration and drying remembers sample 3.
It by the testing result to three batches of samples, finds in enzymolysis process, if being added without any protective agent, sample is living
Property is lower, and heparin structure is destroyed in enzymolysis process, and the natrium carbonicum calcinatum and 0.3% of 0.3% concentration of liquor capacity is added
2 activity of sample of sodium hydrogensulfite is lower than sample 3, and protectant type and dosage in enzymolysis process has finally been determined.
Case study 4: sample being merged and is dissolved, and adjusts feed liquid temperature to 25.0 DEG C, with 4mol/L sodium hydroxide solution tune
Material liquid pH value is saved to 10, one third feed liquid is taken out, sodium chloride is added by the 2.0% of material liquid volume, material liquid volume 2.0% is added
Hydrogen peroxide (original content 30%) aoxidize 12h, filtering, by material liquid volume: ethyl alcohol volume=1:1.0-1:1.2 is added and has risen
The ethyl alcohol of temperature to 40.0 DEG C controls feed liquid concentration of alcohol 42%, and temperature is 35.0 DEG C, and heat preservation staticly settles 4h, and precipitating dehydration is dry
It is dry, remember sample 4.
Case study 5: the natrium carbonicum calcinatum of material liquid volume 0.3% and 0.3% anhydrous sulphur is added in another one third feed liquid
Sour sodium is added sodium chloride by the 2.0% of material liquid volume, hydrogen peroxide (original content 30%) oxygen of material liquid volume 2.0% is added
Change 12h, filtering, by material liquid volume: ethyl alcohol volume=1:1.0-1:1.2 addition has warmed up to 40.0 DEG C of ethyl alcohol, controls feed liquid
Concentration of alcohol 42%, temperature are 35.0 DEG C, and heat preservation staticly settles 4h, and precipitating dehydration and drying remembers sample 5.
Case study 6: remaining one third feed liquid be added material liquid volume 0.3% natrium carbonicum calcinatum and 0.25% it is anhydrous
Sodium sulphate is added sodium chloride by the 2.0% of material liquid volume, the hydrogen peroxide (original content 30%) of material liquid volume 2.0% is added
12h is aoxidized, filtering, by material liquid volume: ethyl alcohol volume=1:1.0-1:1.2 addition has warmed up to 40.0 DEG C of ethyl alcohol, control material
Liquid concentration of alcohol 42%, temperature are 35.0 DEG C, and heat preservation staticly settles 4h, and precipitating dehydration and drying remembers sample 6.
It by the testing result to three batches of samples, finds in oxidation process, if being added without any protective agent, sample is living
Property is lower, and heparin structure is destroyed in oxidation process；And the natrium carbonicum calcinatum and 0.3% of 0.3% concentration of liquor capacity is added
5 activity of sample of anhydrous sodium sulfate is lower than sample 6, and protectant type and dosage in oxidation process has finally been determined.
Explanation of nouns:
Sausage-casing salt: one of Zigong, Sichuan well mine salt kind is used for livestock products department working process casing.
HP: heparin, since in electrophoresis process, movement speed is slower, also referred to as slowly mobile heparin (SM-H).
DS: dermatan sulfate.
HS: Heparan sulfate, since in electrophoresis process, movement speed is very fast, also referred to as fastly mobile heparin (FM-H).
CS: chondroitin sulfate.
(3) beneficial effect
The present invention provides a kind of preparation processes of high purity heparin sodium.Have compared with prior art following beneficial to effect
Fruit: the preparation process of the high purity heparin sodium, specifically includes the following steps: S1, enzymatic hydrolysis salt solution removing protein: by 10-12 times of heparin
The purified water of sodium crude product weight is warming up to 50.0-60.0 DEG C, and the casing of purifying water volume 2.0%-5.0% is added under stirring
Heparin sodium crude is added in salt after dissolution, continue stirring and dissolving at least 1 hour, after dissolution, feed liquid temperature is down to 35.0-
45.0 DEG C, the pancreatin dissolved is added, stirs evenly, S2, once oxidation: adjusting filtrate temperature to 25.0-40.0 DEG C, use
4mol/L sodium hydroxide solution adjusts material liquid pH value to 10-11, add material liquid volume 0.3% natrium carbonicum calcinatum and
0.25% anhydrous sodium sulfate is added sodium chloride by the 2.0% of material liquid volume, the peroxidating hydrogen-oxygen of material liquid volume 2.0% is added
Change 12-18h, S3, precipitating: after oxidation, filtering feed liquid, by material liquid volume: ethyl alcohol volume 1:1.0-1:1.2 addition has warmed up
To 40.0-50.0 DEG C of ethyl alcohol, feed liquid concentration of alcohol 40 ± 2% is controlled, temperature is 35.0-40.0 DEG C, and heat preservation staticly settles 4
± 1h, pumps supernatant, and impurity in supernatant is sunk again, is tested and analyzed with HPLC method to heavy impurity again, S4, secondary oxidation:
The purified water of 6.9-7.3 times of heparin sodium crude weight is added into oxidation precipitation tank II, adjusts feed liquid temperature to 25.0-30.0
DEG C, adjust material liquid pH value to 10-11 with 4mol/L sodium hydroxide solution, add material liquid volume 0.3% natrium carbonicum calcinatum and
0.25% anhydrous sodium sulfate, stirring and dissolving, then sodium chloride is added by the 2.0% of material liquid volume, material liquid volume 2.0% is added
Hydrogen peroxide oxidation 12-18h, S5, again precipitate: after oxidation, filter feed liquid, by material liquid volume: ethyl alcohol volume=1:
1.1-1:1.3 addition has warmed up to 40.0-50.0 DEG C of ethyl alcohol, controls feed liquid concentration of alcohol 46 ± 2%, temperature 35.0-
40.0 DEG C, heat preservation staticly settles 4 ± 1h, pumps supernatant, impurity in supernatant is sunk again, is examined with HPLC method to heavy impurity again
Analysis is surveyed, S6, freeze-drying: dissolving sediment sufficiently with the purified water of 3 times of heparin sodium crude amounts, with 4mol/L hydrochloric acid by pH
Value is transferred to 6.0 ± 0.5, filters, freeze-drying, and high purity heparin sodium can be obtained, it can be achieved that by taking enzymatic hydrolysis salt solution one
Formula dissociates mode, and in dissociation process, and natrium carbonicum calcinatum and anhydrous sodium sulfite is added and prepares at a specific ratio, in height
In warm dissociation process, heparin is protected not to be damaged, remove different impurities with different concentration ethanol, in oxidation process, is added
Natrium carbonicum calcinatum and anhydrous sodium sulfate, are prepared with special ratios, because producing to heparin sodium structure when solving the purification of existing heparin sodium
Raw destroy and be difficult to the problem of obtaining the heparin sodium of high-purity, while using HPLC method, to the impurity removed in each step into
Row detection, easy to operate, product yield is high, more effectively control final product quality.
Detailed description of the invention
Fig. 1 is positioning schematic diagram of the HPLC method of the present invention to each group swarming；
Fig. 2 is the analysis chart that HPLC method of the present invention prepares that heparin sodium removes impurity；
Fig. 3 is detection peak figure spectrogram of the comparative example HPLC method of the present invention to sample；
Fig. 4 is detection peak value figure of the comparative example HPLC method of the present invention to sample；
Fig. 5 is the experimental result table figure of case study three of the present invention；
Fig. 6 is the experimental result table figure of case study six of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Fig. 1-5 is please referred to, the embodiment of the present invention provides a kind of two kinds of technical solutions: preparation process of high purity heparin sodium,
Specifically include following embodiment:
Embodiment 1
S1, the purified water of 10 times of heparin sodium crude weight is warming up to 50.0 DEG C, purifying water volume is added under stirring
Heparin sodium crude is added in 2.0% sausage-casing salt after dissolution, continue stirring and dissolving 1 hour, is cooled to 35.0 DEG C, and heparin sodium is added
1.2 times of pancreatin dissolved of crude product weight and potency ratio, stir evenly, and control 35.0 DEG C of temperature, the hydrogen of 4mol/L is added dropwise
Sodium hydroxide solution adjusts pH value to 7, keeps the temperature enzyme digestion reaction 3.0h, be added 0.3% concentration of liquor capacity natrium carbonicum calcinatum and
The sodium hydrogensulfite of 0.25% concentration is warming up to 50.0 DEG C, and 2.0% anhydrous calcium chloride of liquor capacity is added, is warming up to 80.0 DEG C,
Insulation reaction 30 minutes, 2.0% natrium carbonicum calcinatum of liquor capacity was added, after insulated and stirred reacts 30min in cooling water temperature filtering
Filtering, while HPLC analysis method is used, the impurity of filtering is analyzed, remembers meifu d.
S2, the solution temperature adjustment for obtaining step S1 to 25.0 DEG C, with 4mol/L sodium hydroxide solution adjust material liquid pH value to
10, the natrium carbonicum calcinatum of material liquid volume 0.3% and 0.25% anhydrous sodium sulfate are added, is added by the 2.0% of material liquid volume
Sodium chloride, the hydrogen peroxide (original content 30%) that material liquid volume 2.0% is added aoxidize 12h.
S3, the solution for obtaining step S2 filter, by material liquid volume: ethyl alcohol volume=1:1.0 addition has warmed up to 40.0
DEG C ethyl alcohol, control feed liquid alcoholic strength 38%, temperature is 35.0 DEG C, and heat preservation staticly settles 3.0h, while supernatant answered it is heavy,
Multiple hypostasis is that the impurity removed tests and analyzes impurity with HPLC analysis method, remembers 1fu.d.
S4,6.9 times of the purified water of precipitating that step S3 is obtained is dissolved, adjusts feed liquid temperature to 25.0 DEG C, uses 4mol/
L sodium hydroxide solution adjusts material liquid pH value to 11, adds the natrium carbonicum calcinatum of material liquid volume 0.3% and 0.25% anhydrous sulphur
Sour sodium, stirring and dissolving, then sodium chloride is added by the 2.0% of material liquid volume, the hydrogen peroxide that material liquid volume 2.0% is added is (former dense
Degree is 30%) oxidation 12h.
S5, the solution for obtaining step 4 filter, by material liquid volume: ethyl alcohol volume=1:1.1 addition has warmed up to 40.0 DEG C
Ethyl alcohol, control feed liquid alcoholic strength 44%, temperature is 35.0 DEG C, and heat preservation staticly settles 3.0h, while supernatant answered it is heavy, it is multiple heavy
Object is that the impurity removed tests and analyzes impurity with new analysis method, remembers 2.3fu.d.
S6, freeze-drying: keep sediment sufficiently molten the purified water for 3 times of the precipitating heparin sodium crude amounts that step 7 obtains
PH value is transferred to 5.5 with 4mol/L hydrochloric acid by solution, and filtering, freeze-drying obtain heparin sodium finished product, remember 3Chen.d.
Embodiment 2
S1, the purified water of 12 times of heparin sodium crude weight is warming up to 60.0 DEG C, purifying water volume is added under stirring
Heparin sodium crude is added in 5.0% sausage-casing salt after dissolution, continue stirring and dissolving 1.5 hours, is cooled to 45.0 DEG C, and heparin is added
1.8 times of pancreatin dissolved of sodium crude product weight and potency ratio, stir evenly, and control 45.0 DEG C of temperature, are added dropwise 4mol/L's
Sodium hydroxide solution adjusts pH value to 9, keeps the temperature enzyme digestion reaction 5h, add material liquid volume 0.3% natrium carbonicum calcinatum and
0.25% sodium hydrogensulfite is warming up to 70.0 DEG C, and 3.0% anhydrous calcium chloride of liquor capacity is added, and is warming up to 90.0 DEG C, heat preservation
Reaction 60 minutes is added 3.0% natrium carbonicum calcinatum of liquor capacity, filters after insulated and stirred reaction 60min.
S2, the solution for obtaining step 1, adjust the temperature to 40.0 DEG C, adjust material liquid pH with 4mol/L sodium hydroxide solution
Value adds the natrium carbonicum calcinatum of material liquid volume 0.3% and 0.25% anhydrous sodium sulfate, by the 2.0% of material liquid volume to 11
Sodium chloride is added, the hydrogen peroxide (concentration of hydrogen peroxide 30%) that material liquid volume 2.0% is added aoxidizes 18h.
S3, the solution for obtaining step 2 filter, by material liquid volume: ethyl alcohol volume=1:1.2 addition has warmed up to 50.0 DEG C
Ethyl alcohol, control feed liquid concentration of alcohol 42%, temperature be 40.0 DEG C, heat preservation staticly settle 5h.
S4, the purified water for 7.3 times of weight of precipitating that step 3 obtains is dissolved, adjusts feed liquid temperature to 30.0 DEG C, uses
4mol/L sodium hydroxide solution adjusts material liquid pH value to 11, adds the natrium carbonicum calcinatum and 0.25% of material liquid volume 0.3%
Anhydrous sodium sulfate stirring and dissolving, then sodium chloride is added by the 2.0% of material liquid volume, the hydrogen peroxide of material liquid volume 2.0% is added
(concentration of hydrogen peroxide 30%) aoxidizes 18h.
S5, the solution for obtaining step 4 filter, by material liquid volume: ethyl alcohol volume=1:1.3 addition has warmed up to 50.0 DEG C
Ethyl alcohol, control feed liquid concentration of alcohol 48%, temperature be 40.0 DEG C, heat preservation staticly settle 5h.
S6, freeze-drying: the purified water that the precipitating that step 5 is obtained is measured with 3 times sufficiently dissolves, will with 4mol/L hydrochloric acid
PH value is transferred to 6.5, and filtering, freeze-drying obtain heparin sodium finished product, remember dongganChen.d.
Described above, as the embodiment of the present invention compares, liver prepared by the present invention with other inventive method samples
Plain sodium, is free of other magazines, sample purity highest, and attached drawing 2 is to digest salt solution by test map and positioning map comparative analysis
Impurity note-the meifu.d that process removes is CS, and once oxidation precipitating is controlled, the impurity note -1fu.d of removing by alcoholic strength,
For a large amount of DS, a small amount of HS, CS；Secondary oxidation precipitating is controlled by alcoholic strength, and it is a large amount of that the impurity of removing, which remembers 23fu.d,
HS and a small amount of DS.
Comparative example: patent (CN201810189164) inventive embodiments
1) it dissolves: heparin sodium crude being dissolved in 3.5% sodium chloride solution, and be heated to 50 DEG C, with NaOH solution tune
PH to 8.2 is saved, is filtered after standing 2.5h, insoluble impurities is removed, obtains clear filtrate；
2) it digests: protease being added in the clear filtrate described in step 1), adjust pH to 8, temperature control is existed
42 DEG C, enzymolysis time 5.5h；
3) it adjusts acid: sodium hydrogensulfite is added as protective agent, addition salt acid for adjusting pH to 1.8 maintains temperature at 21 DEG C, from
Heart deproteination；
4) it aoxidizes: hydrogen peroxide, the addition of hydrogen peroxide is added in the filtrate of the centrifugation deproteination described in step 3)
Amount is the 2.5% of filtrate quality, adjusts pH to 8.8, and temperature is controlled at 26 DEG C, oxidation reaction 14.5h is carried out；
5) precipitate: salt acid for adjusting pH being added in the liquid that oxidation reaction described in step 4) is completed to 6.5, be added with
95% ethyl alcohol of liquid aliquot is precipitated, sedimentation time 12.5h, is extracted supernatant out, is obtained sediment, and sediment is slow
Slowly be added in acetone be dehydrated it is secondary；
6) dry: by dewatered sediment described in step 5) at 58 DEG C vacuum drying 5h, obtain exquisite heparin sodium
Remember 2Chen.d.
Further, the ratio of heparin sodium crude and sodium chloride is 1:14 in step 1).
Further, the additional amount of protease described in step 2) is the 0.08% of filtrate quality.
This example is the preparation method under patent (CN201810189164), and prepared color sample is partially yellow, and is used
To acetone, it is unfavorable for mass production, and its sample is detected, containing other impurities, sample purity is not high, such as attached drawing 3 and attached
Described in Fig. 4.
By being compared to the present invention and the sample quality of patent (CN201810189164), can clearly find, sample
The sample of 2Chen.d, that is, patent (CN201810189164) has the impurity peaks of very little, and impurity is Heparan sulfate, and sample
3Chen.d and dongganChen.d is the sample of two examples preparation of the invention, only one heparin peak is free of other impurities,
The present invention is primarily due in enzymolysis process, using the natrium carbonicum calcinatum of special ratios and sodium hydrogensulfite as protective agent, no
Only make to maintain stable alkaline nature under high temperature, so that albumen is thoroughly denaturalized, and will not destroy heparin sodium structure, in oxygen again
During change, equally using the natrium carbonicum calcinatum of special ratios and anhydrous sodium sulfate as protective agent, in oxidation process, not only hold
It is continuous provide oxidation needed for alkaline condition so that the steady and sustained progress of oxidation effectiveness, oxidation effectiveness is more preferably, while anhydrous
The addition of sodium sulphate provides a large amount of sulfate ion in oxidation process, is ensured that in this way even if more violent oxidation
Also heparin structure can not be destroyed, oxidation is more abundant, and secondly we are by fractional precipitation, with different precipitating alcoholic strengths, to difference
Impurity is removed, and under lower alcohol concentration, is eliminated dermatan sulfate, is improved alcohol concentration, removes acetyl sulfate liver
Element ensures that the high-purity of product in this way, and therefore, the heparin sodium of production method preparation, is free of other impurities through the invention,
Sample purity highest, 2Chen.d is the sample that under patent (CN201810189164) prepared by preparation method in attached drawing 3,
3Chen.d and dongganChen.d is the sample of two examples preparation of the invention, and 2Chen.d is patent in attached drawing 4
(CN201810189164) sample that under prepared by preparation method, 3Chen.d and dongganChen.d are two example systems of the invention
Standby sample.
It should be noted that, in this document, relational terms such as first and second and the like are used merely to a reality
Body or operation are distinguished with another entity or operation, are deposited without necessarily requiring or implying between these entities or operation
In any actual relationship or order or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to
Non-exclusive inclusion, so that the process, method, article or equipment including a series of elements is not only wanted including those
Element, but also including other elements that are not explicitly listed, or further include for this process, method, article or equipment
Intrinsic element.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. a kind of preparation process of high purity heparin sodium, it is characterised in that: specifically includes the following steps:
S1, enzymatic hydrolysis salt solution removing protein: the purified water of 10-12 times of heparin sodium crude weight is warming up to 50.0-60.0 DEG C, stirs shape
The sausage-casing salt of purifying water volume 2.0%-5.0% is added under state, heparin sodium crude is added after dissolution, continues stirring and dissolving at least 1
Hour, after dissolution, feed liquid temperature is down to 35.0-45.0 DEG C, the pancreatin dissolved is added, stirs evenly, controls temperature
35.0-45.0 DEG C, pH value is adjusted to 7-9 with sodium hydroxide solution, enzyme digestion reaction 3.0-5.0 hours is kept the temperature, material liquid volume is added
0.3% natrium carbonicum calcinatum and 0.25% sodium hydrogensulfite, be warming up to 50.0-70.0 DEG C, the anhydrous chlorination dissolved be added
Calcium is warming up to 80.0-90.0 DEG C, insulation reaction 30-60 minutes, natrium carbonicum calcinatum is added, after insulated and stirred is reacted 30-60 minutes
Filtering tests and analyzes filtering removal impurity with HPLC method；
S2, once oxidation: adjust filtrate temperature to 25.0-40.0 DEG C, with 4mol/L sodium hydroxide solution adjust material liquid pH value to
10-11 adds the natrium carbonicum calcinatum of material liquid volume 0.3% and 0.25% anhydrous sodium sulfate, adds by the 2.0% of material liquid volume
Enter sodium chloride, the hydrogen peroxide oxidation 12-18h of material liquid volume 2.0% is added；
S3, precipitating: after oxidation, filter feed liquid, by material liquid volume: ethyl alcohol volume 1:1.0-1:1.2 be added have warmed up to
40.0-50.0 DEG C of ethyl alcohol controls feed liquid concentration of alcohol 40 ± 2%, and temperature is 35.0-40.0 DEG C, and heat preservation staticly settles 4 ±
1h pumps supernatant, and impurity in supernatant is sunk again, is tested and analyzed with HPLC method to heavy impurity again；
S4, secondary oxidation: the purified water of 6.9-7.3 times of heparin sodium crude weight being added into oxidation precipitation tank II, adjusts feed liquid
Temperature adjusts material liquid pH value to 10-11 to 25.0-30.0 DEG C, with 4mol/L sodium hydroxide solution, adds material liquid volume 0.3%
Natrium carbonicum calcinatum and 0.25% anhydrous sodium sulfate, stirring and dissolving, then by material liquid volume 2.0% be added sodium chloride, be added
The hydrogen peroxide oxidation 12-18h of material liquid volume 2.0%；
S5, it precipitates again: after oxidation, filtering feed liquid, by material liquid volume: ethyl alcohol volume=1:1.1-1:1.3 addition has risen
The ethyl alcohol of temperature to 40.0-50.0 DEG C controls feed liquid concentration of alcohol 46 ± 2%, and temperature is 35.0-40.0 DEG C, and heat preservation staticly settles 4
± 1h, pumps supernatant, and impurity in supernatant is sunk again, is tested and analyzed with HPLC method to heavy impurity again；
S6, freeze-drying: the purified water measured with 3 times of heparin sodium crude dissolves sediment sufficiently, with 4mol/L hydrochloric acid by pH value
6.0 ± 0.5 are transferred to, is filtered, high purity heparin sodium can be obtained in freeze-drying.
2. a kind of preparation process of high purity heparin sodium according to claim 1, it is characterised in that: pancreas in the step S1
The ratio of protease is 1.2-1.8 times of crude product weight and potency ratio.
3. a kind of preparation process of high purity heparin sodium according to claim 1, it is characterised in that: nothing in the step S1
Water calcium chloride concentration is 2.0%-3.0%.
4. a kind of preparation process of high purity heparin sodium according to claim 1, it is characterised in that: nothing in the step S1
Aqueous sodium carbonate concentration is 2.0%-3.0%.
5. a kind of preparation process of high purity heparin sodium according to claim 1, it is characterised in that: the step S2 and step
The original content of hydrogen peroxide is 30% in rapid S4.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112194740A (en) * | 2020-10-23 | 2021-01-08 | 宋江 | Method for extracting heparin sodium from small intestine of pig |
| CN113735995A (en) * | 2020-05-29 | 2021-12-03 | 江苏唯高生物科技有限公司 | Novel process for preparing heparin sodium crude product |
| CN115028757A (en) * | 2022-06-29 | 2022-09-09 | 江苏麦德森制药有限公司 | Decolorizing method of heparin sodium |
| TWI796957B (en) * | 2022-02-18 | 2023-03-21 | 蒂美生技股份有限公司 | Method for extracting heparin |
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