CN107056966A - A kind of process for purification of liquaemin - Google Patents
A kind of process for purification of liquaemin Download PDFInfo
- Publication number
- CN107056966A CN107056966A CN201710288789.9A CN201710288789A CN107056966A CN 107056966 A CN107056966 A CN 107056966A CN 201710288789 A CN201710288789 A CN 201710288789A CN 107056966 A CN107056966 A CN 107056966A
- Authority
- CN
- China
- Prior art keywords
- liquaemin
- purification
- nacl solution
- solution
- enzymolysis liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of process for purification of liquaemin, it is related to the preparing technical field of liquaemin.The process for purification of liquaemin includes：Crude heparin sodium is dissolved in NaCl solution and obtains thick solution；The thick solution successively digest twice and obtains enzymolysis liquid；Precipitation is filtered to take after ethanol, agitated standing are added into the enzymolysis liquid；Upper ion exchange column purification obtains eluent after the precipitation is completely dissolved；Absorption chromatograph column on the eluent is decolourized to obtain heparin sodium aqua.The process for purification for the liquaemin that the present invention is provided has the simple to operate, process time short, the characteristics of product yield is high, and products obtained therefrom absorptivity is low, and purity is high, and activity is high.
Description
Technical field
The present invention relates to the preparing technical field of liquaemin, especially a kind of process for purification of liquaemin.
Background technology
Liquaemin is a kind of acidic mucopolysaccharide, with anticoagulant effect.Liquaemin as a kind of natural anticoagulative substance,
In anticoagulation, promote that there is good activity in terms of lipoprotein lipase release and the molten cell system of complement, be widely used in popularity
Encephalitis, septicemia, blood embolization, acute myocardial infarction, artery sclerosis etc. are treated.
The bulk drug of liquaemin derives from heparin crude product.Heparin crude product is derived from the mucous membrane of small intestine of healthy animal, wherein containing
A large amount of foreign protein, heteronuclear acid, microorganisms etc., need to pass through physics and chemical extraction separation process, and orientation obtains natural structure group
Complete heparin, so that liquaemin bulk drug is made.
Existing heparin method for extraction and purification removes the materials such as impurity protein, nucleic acid using soda acid processing mostly, using multiple
The method of oxidation removes colors, the heteroglycan such as removes by the way of ethanol multiple fractionation.The side of above-mentioned soda acid processing
Formula easily causes the residual quantity height of nucleic acid, pigment, causes subsequent oxidation increased frequency；Acid treatment and repeatedly oxidation are easily caused liquaemin
Partial inactivation, it is difficult to obtain the liquaemin of high-titer；Multiple alcohol grading easily causes yield reduction and the waste of material.
The content of the invention
The goal of the invention of the present invention is：For above-mentioned problem, there is provided a kind of process for purification of liquaemin, the party
Method has the simple to operate, process time short, the characteristics of product yield is high, and products obtained therefrom absorptivity is low, and purity is high, activity
It is high.
The technical solution adopted by the present invention is as follows：
A kind of process for purification of liquaemin, it includes：
Dissolving crude product：Crude heparin sodium is dissolved in NaCl solution and obtains thick solution；
Digest twice：The thick solution successively digest twice and obtains enzymolysis liquid；
Ethanol precipitation：Precipitation is filtered to take after ethanol, agitated standing are added into the enzymolysis liquid；
Ion exchange column purification：Upper ion exchange column purification obtains eluent after the precipitation is completely dissolved；
Absorption chromatograph column decolourizes：Absorption chromatograph column on the eluent is decolourized to obtain heparin sodium aqua.
Heparin is combined into compound with protein in vivo, and the anticoagulant of compound increases with the removal of its protein active
By force.In this method, crude heparin sodium by proteolysis twice, by liquaemin from protein complex abundant separate out,
Protein residues are avoided to influence its activity.Enzymolysis liquid avoids precipitating using ethanol multiple fractionation, can effectively improve product yield, drop
Low material waste.Liquaemin first passes through ion exchange column preliminary purification, except impurity such as foreigh protein removing and heteroglycan, then by inhaling
Attached chromatographic column decolourizes, it is to avoid oxidative decoloration, so as to avoid introducing foreign matter and heparin sodium inactivation, improves the activity of liquaemin.Together
When, the purity of product is ensure that using ion exchange column purification and absorption chromatograph column decolouring two-step purifying.
In invention preferred embodiment, during dissolving crude product, NaCl solution is heated to 30-45 DEG C, the NaCl is molten
The concentration of liquid is 2%（m/v）, the ratio between the crude heparin sodium and the NaCl solution are 1：5-10（kg：L）, and at 30-45 DEG C
Lower holding 4-6h.
The temperature of NaCl solution is improved, liquaemin solubility wherein and rate of dissolution can be improved, so as to improve thing
The utilization rate of material.
In preferred embodiments of the present invention, enzymolysis includes the first enzymolysis and the second enzymolysis twice；First enzymolysis includes：Regulation
The thick solution is 7.5-8.5 to pH and is heated to 45-50 DEG C, and it is 0.5% then to add protease I to its mass percent, molten
Liquid pH maintains 4-6h to obtain the first enzymolysis liquid for holding under the conditions of 7.5-8.5,45-50 DEG C of temperature.
In preferred embodiments of the present invention, adjust first enzymolysis liquid and be 8.5-9.5 to pH and be heated to 55-60 DEG C,
Then add Protease III to its mass percent be 0.5%, pH value of solution be 8.5-9.5,55-60 DEG C of temperature under the conditions of keep 4-
6h, adjusts pH to 6.5-7.0 and obtains enzymolysis liquid after being heated to 85-90 DEG C, insulation 0.5h.
Digested due to taking the protein in above-mentioned technical proposal, protein complex, liquaemin therein is abundant
Ground separate out, and pass through high temperature degradation again into the LMWHs of high added value.
In preferred embodiments of the present invention, ethanol precipitation process includes adding 1.5 times of enzymolysis liquid into the enzymolysis liquid
95% long-pending ethanol, 4h-6h is staticly settled after stirring；Filter to take precipitation.
Due to taking above-mentioned technical proposal, liquaemin is precipitated in ethanol, effectively separates liquaemin by filtering,
Meanwhile, significantly reduce material waste without ethanol multiple fractionation precipitation.
In preferred embodiments of the present invention, ion exchange column purification includes：By it is described precipitation be completely dissolved after, regulation pH to
7.5-8.0, is warming up to upper prop after 45-50 DEG C, and upper prop speed is to balance 1h after the completion of 1.0BV/h, upper prop, is then successively washed
Removing impurities matter and elution liquaemin.
In preferred embodiments of the present invention, with the distillation water washing that 2BV temperature is 40-50 DEG C, flow velocity is 2BV/h；Respectively
Washed twice with 2BV 1.0 mol/L NaCl solution, flow velocity is 1.5BV/h, for the first time regulation NaCl solution pH to 5.0-
5.5, second of regulation NaCl solution pH 9.5-10.0；Washed again with 2BV 1.5 mol/L NaCl solution, flow velocity is
0.5BV/h；Elution liquaemin includes：Eluted with the NaCl solution that 2BV temperature is 40-50 DEG C, concentration is 2.5-3.5 mol/L,
Flow velocity is 0.5BV/h.
Ion exchange column purification is the preliminary purification to heparin sodium, and ion exchange column packing is that D254 strong basicities quaternary ammonium type is cloudy
Ion exchange resin, it can have high efficiency adsorbing liquaemin, it is ensured that liquaemin is adsorbed completely.Eluting impurity includes elution small molecule
Impurity-distillation water washing, 1.0 mol/L NaCl solution are washed twice；Elute the mol/L of heteroglycan -1.5 NaCl solution
Washing.
In preferred embodiments of the present invention, absorption chromatograph column decolourizes to include：By the eluent upper prop, balance after 1h successively
Carry out washing decolouring and elution liquaemin.
In preferred embodiments of the present invention, washing decolouring includes：With the distillation water washing that 2BV temperature is 40-50 DEG C, flow velocity
For 2BV/h.
In preferred embodiments of the present invention, elution liquaemin includes：Be 40-50 DEG C with 2BV temperature, concentration be 0.5 mol/
L NaCl solution elution, flow velocity is 0.5BV/h.
Absorption chromatograph column decolourizes to be that, to the secondarily purified of liquaemin, its main purpose is to remove depigmentaton.Absorption chromatograph column
Filler is D208 macroporous absorbent resins, and the resin can efficiently separate liquaemin and pigment, reduce the absorptivity of product.The party
In case, first adsorption column is washed with distilled water, the lower pigment of elution, distilled water is heated into 40-50 DEG C can effectively improve color
The eluting rate of element, then elutes liquaemin with the NaCl solution that temperature is 40-50 DEG C, concentration is 0.5 mol/L, obtains high-purity
Liquaemin.
In summary, by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
（1）Crude heparin sodium by proteolysis twice, by liquaemin from protein complex abundant separate out, it is to avoid egg
Its activity of white matter remaining influence.
（2）Enzymolysis liquid avoids precipitating using ethanol multiple fractionation, improves product yield, reduction material waste.
（3）Liquaemin decolourizes to purify twice altogether respectively through ion exchange column preliminary purification, absorption chromatograph column, obtained production
Product purity is high, and avoids and use oxidative decoloration, so that introducing foreign matter and heparin sodium inactivation are avoided, so as to improve heparin
The activity of sodium.
Embodiment
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
This specification（Including any accessory claim, summary）Disclosed in any feature, unless specifically stated otherwise,
Replaced by other equivalent or with similar purpose alternative features.I.e., unless specifically stated otherwise, each feature is a series of
An example in equivalent or similar characteristics.
Embodiment 1
The present embodiment provides a kind of process for purification of liquaemin, and it includes：
1. dissolving crude product：Weigh crude heparin sodium 100kg to be placed in stainless steel cask, add 500L 2%（m/v）NaCl solution,
It is heated to 30 DEG C, heat-insulation soaking 6h makes crude heparin sodium fully dissolve to obtain thick solution and vacuum is pumped into enzymatic vessel.
2. digest twice：In enzymatic vessel, thick solution is adjusted into pH to 7.5, and is warming up to 45 DEG C, addition protease I
2.5kg, it is that 7.5, temperature is to stir and be incubated 6h under the conditions of 45 DEG C to obtain the first enzymolysis liquid to keep pH value of solution.Regulation first is digested
The pH of liquid is warming up to 55 DEG C to 8.5, adds 0.5% Protease III 2.5kg, and it is that 8.5, temperature is 55- DEG C of condition to keep pH value of solution
Lower stir simultaneously is incubated 6h, then adjusts pH to 6.5 and is warming up to 85 DEG C, is incubated 0.5h, obtains enzymolysis liquid.
3. centrifugation and filtering：Enzymolysis liquid is cooled to less than 60 DEG C, 2h is centrifuged under the conditions of 4000rpm, centrifugate vacuum is inhaled
Enter fluid reservoir, and stainless steel sheet frame is depressed into from fluid reservoir vacuum and be decompressed to settling tank again, filtering to obtaining filtrate clearly.
4. ethanol precipitation：Lower 95% ethanol for adding 1.5 times of amounts of above-mentioned filtrate stirring is taken, after stirring, is staticly settled
Precipitation is taken after 4h, vacuum filtration.
5. dissolving：Take above-mentioned precipitation to add 500L purified waters stirring 2h, be completely dissolved and obtain solution.
6.D254 ion exchange column purifications：Above-mentioned solution is adjusted into pH to 7.5,45 DEG C are warming up to, then will with infusion pump
Solution inputs ion exchange column, and exchange velocity is 1.0BV/h, and the mother liquor after exchange is squeezed into essence and irrigated by lifting water to a higher level with a water pump, etc., heparin in detection efflux
Content, if also heparin, by essence irrigate by lifting water to a higher level with a water pump, etc. in solution squeeze into ion exchange column again, efflux squeezes into settling tank again, extremely
During no-rod tractor, stop ion exchange, balance 1h.
7. wash small molecular weight impurity：40 DEG C of the distillation water washing of the ion exchange column that finishes with 2BV will be balanced, flow velocity is
2BV/h, then washed 2 times with 2BV 1.0 mol/L NaCl, flow velocity is 1.5BV/h.For the first time regulation NaCl solution pH to
5.0, the second regulation NaCl solution pH to 9.5.
8. elute heteroglycan：First with the mol/L NaCl solutions of 2BV 1.5 elution heteroglycan, flow velocity is 0.5BV/h.
9. elute liquaemin：Ion exchange column is eluted into heparin, flow velocity with 40 DEG C, 2BV, 2.5 mol/L NaCl solution
For 0.5BV/h, collect efflux and obtain eluent.
10.D208 absorption chromatograph columns decolourize：Eluent is inputted into absorption chromatograph column with infusion pump, upper prop speed is 1.0BV/
1h is balanced after the completion of h, upper prop, is decolourized with 2BV temperature for 40 DEG C of distillation water washing, flow velocity is 2BV/h.After the completion of decolouring, use
The NaCl solution that 2BV temperature is 40 DEG C, concentration is 0.5 mol/L affords heparin sodium aqua, and flow velocity is 0.5BV/h.
11. concentrated compounding and micro porous filtration：95% ethanol that 0.7 times of volume is added into above-mentioned heparin sodium aqua is precipitated, and is sunk
Suction filtration goes precipitation after the 2h of shallow lake, precipitation is dissolved into 15% concentration with distilled water, with taking filtrate after 0.45um filtering with microporous membrane.
12. lyophilized and crushing：Above-mentioned filtrate is placed in freeze dryer and freezed, crushes and 60 mesh sieves excessively obtains fine work heparin
Sodium.
Embodiment 2
The present embodiment provides a kind of process for purification of liquaemin, and it includes：
1. dissolving crude product：Weigh crude heparin sodium 100kg to be placed in stainless steel cask, add 500L 2%（m/v）NaCl solution,
It is heated to 38 DEG C, heat-insulation soaking 5h makes crude heparin sodium fully dissolve to obtain thick solution and vacuum is pumped into enzymatic vessel.
2. digest twice：In enzymatic vessel, thick solution is adjusted into pH to 8.0, and is warming up to 47 DEG C, addition protease I
2.5kg, it is that 8.0, temperature is to stir and be incubated 5h under the conditions of 47 DEG C to obtain the first enzymolysis liquid to keep pH value of solution.Regulation first is digested
The pH of liquid is warming up to 58 DEG C to 9.0, adds 0.5% Protease III 2.5kg, and it is that 9.0, temperature is 58 DEG C of conditions to keep pH value of solution
Lower stir simultaneously is incubated 5h, then adjusts pH to 6.8 and is warming up to 88 DEG C, is incubated 0.5h, obtains enzymolysis liquid.
3. centrifugation and filtering：Enzymolysis liquid is cooled to less than 60 DEG C, 2h is centrifuged under the conditions of 4000rpm, centrifugate vacuum is inhaled
Enter fluid reservoir, and stainless steel sheet frame is depressed into from fluid reservoir vacuum and be decompressed to settling tank again, filtering to obtaining filtrate clearly.
4. ethanol precipitation：Lower 95% ethanol for adding 1.5 times of amounts of above-mentioned filtrate stirring is taken, after stirring, is staticly settled
Precipitation is taken after 4h, vacuum filtration.
5. dissolving：Take above-mentioned precipitation to add 500L purified waters stirring 2h, be completely dissolved and obtain solution.
6.D254 ion exchange column purifications：Above-mentioned solution is adjusted into pH to 7.7,47 DEG C are warming up to, then will with infusion pump
Solution inputs ion exchange column, and exchange velocity is 1.0BV/h, and the mother liquor after exchange is squeezed into essence and irrigated by lifting water to a higher level with a water pump, etc., heparin in detection efflux
Content, if also heparin, by essence irrigate by lifting water to a higher level with a water pump, etc. in solution squeeze into ion exchange column again, efflux squeezes into settling tank again, extremely
During no-rod tractor, stop ion exchange, balance 1h.
7. wash small molecular weight impurity：47 DEG C of the distillation water washing of the ion exchange column that finishes with 2BV will be balanced, flow velocity is
2BV/h, then washed 2 times with 2BV 1.0 mol/L NaCl, flow velocity is 1.5BV/h.For the first time regulation NaCl solution pH to
5.2, the second regulation NaCl solution pH to 9.7.
8. elute heteroglycan：First with the mol/L NaCl solutions of 2BV 1.5 elution heteroglycan, flow velocity is 0.5BV/h.
9. elute liquaemin：Ion exchange column is eluted into heparin, flow velocity with 45 DEG C, 2BV, 3.0 mol/L NaCl solution
For 0.5BV/h, collect efflux and obtain eluent.
10.D208 absorption chromatograph columns decolourize：Eluent is inputted into absorption chromatograph column with infusion pump, upper prop speed is 1.0BV/
1h is balanced after the completion of h, upper prop, is decolourized with 2BV temperature for 45 DEG C of distillation water washing, flow velocity is 2BV/h.After the completion of decolouring, use
The NaCl solution that 2BV temperature is 45 DEG C, concentration is 0.5 mol/L affords heparin sodium aqua, and flow velocity is 0.5BV/h.
11. concentrated compounding and micro porous filtration：95% ethanol that 0.7 times of volume is added into above-mentioned heparin sodium aqua is precipitated, and is sunk
Suction filtration goes precipitation after the 2h of shallow lake, precipitation is dissolved into 15% concentration with distilled water, with taking filtrate after 0.45um filtering with microporous membrane.
12. lyophilized and crushing：Above-mentioned filtrate is placed in freeze dryer and freezed, crushes and 60 mesh sieves excessively obtains fine work heparin
Sodium.
Embodiment 3
The present embodiment provides a kind of process for purification of liquaemin, and it includes：
1. dissolving crude product：Weigh crude heparin sodium 100kg to be placed in stainless steel cask, add 500L 2%（m/v）NaCl solution,
It is heated to 45 DEG C, heat-insulation soaking 4h makes crude heparin sodium fully dissolve to obtain thick solution and vacuum is pumped into enzymatic vessel.
2. digest twice：In enzymatic vessel, thick solution is adjusted into pH to 8.5, and is warming up to 50 DEG C, addition protease I
2.5kg, it is that 8.5, temperature is to stir and be incubated 4h under the conditions of 50 DEG C to obtain the first enzymolysis liquid to keep pH value of solution.Regulation first is digested
The pH of liquid is warming up to 60 DEG C to 9.5, adds 0.5% Protease III 2.5kg, and it is that 9.5, temperature is 60 DEG C of conditions to keep pH value of solution
Lower stir simultaneously is incubated 4h, then adjusts pH to 7.0 and is warming up to 90 DEG C, is incubated 0.5h, obtains enzymolysis liquid.
3. centrifugation and filtering：Enzymolysis liquid is cooled to less than 60 DEG C, 2h is centrifuged under the conditions of 4000rpm, centrifugate vacuum is inhaled
Enter fluid reservoir, and stainless steel sheet frame is depressed into from fluid reservoir vacuum and be decompressed to settling tank again, filtering to obtaining filtrate clearly.
4. ethanol precipitation：Lower 95% ethanol for adding 1.5 times of amounts of above-mentioned filtrate stirring is taken, after stirring, is staticly settled
Precipitation is taken after 4h, vacuum filtration.
5. dissolving：Take above-mentioned precipitation to add 500L purified waters stirring 2h, be completely dissolved and obtain solution.
6.D254 ion exchange column purifications：Above-mentioned solution is adjusted into pH to 8.0,0 DEG C is warming up to, then will be molten with infusion pump
Liquid inputs ion exchange column, and exchange velocity is 1.0BV/h, and the mother liquor after exchange is squeezed into essence and irrigated by lifting water to a higher level with a water pump, etc., heparin in detection efflux
Content, if also heparin, the solution in essence is irrigated by lifting water to a higher level with a water pump, etc. squeezes into ion exchange column again, and efflux squeezes into settling tank again, to nothing
During heparin, stop ion exchange, balance 1h.
7. wash small molecular weight impurity：50 DEG C of the distillation water washing of the ion exchange column that finishes with 2BV will be balanced, flow velocity is
2BV/h, then washed 2 times with 2BV 1.0 mol/L NaCl, flow velocity is 1.5BV/h.For the first time regulation NaCl solution pH to
5.5, the second regulation NaCl solution pH to 10.0.
8. elute heteroglycan：First with the mol/L NaCl solutions of 2BV 1.5 elution heteroglycan, flow velocity is 0.5BV/h.
9. elute liquaemin：Ion exchange column is eluted into heparin, flow velocity with 50 DEG C, 2BV, 3.5 mol/L NaCl solution
For 0.5BV/h, collect efflux and obtain eluent.
10.D208 absorption chromatograph columns decolourize：Eluent is inputted into absorption chromatograph column with infusion pump, upper prop speed is 1.0BV/
1h is balanced after the completion of h, upper prop, is decolourized with 2BV temperature for 50 DEG C of distillation water washing, flow velocity is 2BV/h.After the completion of decolouring, use
The NaCl solution that 2BV temperature is 50 DEG C, concentration is 0.5 mol/L affords heparin sodium aqua, and flow velocity is 0.5BV/h.
11. concentrated compounding and micro porous filtration：95% ethanol that 0.7 times of volume is added into above-mentioned heparin sodium aqua is precipitated, and is sunk
Suction filtration goes precipitation after the 2h of shallow lake, precipitation is dissolved into 15% concentration with distilled water, with taking filtrate after 0.45um filtering with microporous membrane.
12. lyophilized and crushing：Above-mentioned filtrate is placed in freeze dryer and freezed, crushes and 60 mesh sieves excessively obtains fine work heparin
Sodium.
Embodiment 4
Present embodiments provide the physicochemical property inspection for the refined heparin sodium extracted according to the process for purification of liquaemin in embodiment 1-3
Survey.
Experimental method：
1. product yield inspection
Experimental method：Weighed after the refined heparin sodium obtained according to 3 kinds of process for purification is dried, according to the following formula calculated yield：
Yield（%）=（Fine work weight/crude product weight）×100%
Product yield result is as shown in table 1.
2. absorption chromatograph column decoloration process is verified
Experimental method：The refined heparin sodium prepared according to 3 kinds of process for purification is taken, adds water and 4mg/mL solution is made, determine respectively
, measurement result is as shown in table 2 for absorbance at 260nm, 280nm, 400nm, 420nm, 470nm wavelength.
The yield the result of the process for purification of the liquaemin of table 1
| Group | Theoretical yield scope | Fine work yield (kg) | Actual recovery |
| Embodiment 1 | 60%-80% | 67.4 | 67.4% |
| Embodiment 2 | 60%-80% | 71.2 | 71.2% |
| Embodiment 3 | 60%-80% | 69.3 | 69.3% |
As shown in Table 1, the yield of the finished product obtained according to the process for purification of the liquaemin provided in embodiment 1-3 meets theoretical receipts
Rate scope, thus illustrates, the process for purification feasible process for the liquaemin that the present invention is provided.
The absorption chromatograph column decoloration process the result table of table 2
As shown in Table 2, the finished product obtained according to the process for purification of the liquaemin provided in embodiment 1-3 is inhaled in the light that individual wavelength goes out
Receipts are green to meet standard, thus illustrates that the absorption chromatograph column decoloration process in the process for purification for the liquaemin that the present invention is provided has
Good decolorizing effect.
The invention is not limited in foregoing embodiment.The present invention, which is expanded to, any in this manual to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
Claims (10)
1. a kind of process for purification of liquaemin, it is characterised in that it includes：
Dissolving crude product：Crude heparin sodium is dissolved in NaCl solution and obtains thick solution；
Digest twice：The thick solution successively digest twice and obtains enzymolysis liquid；
Ethanol precipitation：Precipitation is filtered to take after ethanol, agitated standing are added into the enzymolysis liquid；
Ion exchange column purification：Upper ion exchange column purification obtains eluent after the precipitation is completely dissolved；
Absorption chromatograph column decolourizes：Absorption chromatograph column on the eluent is decolourized to obtain heparin sodium aqua.
2. the process for purification of liquaemin according to claim 1, it is characterised in that absorption chromatograph column decolourizes to include：By institute
State eluent upper prop, upper prop speed is to balance 1h after the completion of 1.0BV/h, upper prop, successively carries out washing decolouring and elution heparin
Sodium.
3. the process for purification of liquaemin according to claim 2, it is characterised in that washing decolouring includes：It is with 2BV temperature
40-50 DEG C of distillation water washing, flow velocity is 2BV/h.
4. the process for purification of liquaemin according to claim 2, it is characterised in that elution liquaemin includes：Use 2BV temperature
For the NaCl solution elution that 40-50 DEG C, concentration are 0.5 mol/L, flow velocity is 0.5BV/h.
5. the process for purification of the liquaemin according to any one of claim 1-4, it is characterised in that dissolving crude product process
In, NaCl solution is heated to 30-45 DEG C, the concentration of the NaCl solution is 2%（m/v）, the crude heparin sodium with it is described
The ratio between NaCl solution is 1：5-10（kg：L）, and keep 4-6h at 30-45 DEG C.
6. the process for purification of the liquaemin according to any one of claim 1-4, it is characterised in that enzymolysis includes the twice
One enzymolysis and the second enzymolysis；First enzymolysis includes：Adjust the thick solution to be 7.5-8.5 to pH and be heated to 45-50 DEG C, then
It is 0.5% to add protease I to its mass percent in NaCl solution, and holdings pH value of solution is 7.5-8.5,45-50 DEG C of temperature
Under the conditions of stirring 4-6h obtain the first enzymolysis liquid.
7. the process for purification of liquaemin according to claim 6, it is characterised in that the second enzyme solution includes：Regulation institute
It is 8.5-9.5 and to be heated to 55-60 DEG C that the first enzymolysis liquid, which is stated, to pH, then adds Protease III to its matter in NaCl solution
It is 0.5% to measure percentage, and pH value of solution adjusts pH to 6.5-7.0 and simultaneously added to keep 4-6h under the conditions of 8.5-9.5,55-60 DEG C of temperature
Heat obtains enzymolysis liquid to 85-90 DEG C after insulation 0.5h.
8. the process for purification of the liquaemin according to any one of claim 1-4, it is characterised in that ethanol precipitation process bag
95% ethanol that 1.5 times of enzymolysis liquid volumes are added into the enzymolysis liquid is included, 4h-6h is staticly settled after stirring；Filter to take heavy
Form sediment.
9. the process for purification of the liquaemin according to any one of claim 1-4, it is characterised in that ion exchange column purification
Including：After the precipitation is completely dissolved, pH is to 7.5-8.0 for regulation, is warming up to upper prop after 45-50 DEG C, exchange velocity is
1h is balanced after the completion of 1.0BV/h, upper prop, elution impurity and elution liquaemin is then successively carried out.
10. the process for purification of liquaemin according to claim 9, it is characterised in that elution impurity includes：Use 2BV temperature
For 40-50 DEG C of distillation water washing, flow velocity is 2BV/h；Washed twice respectively with 2BV 1.0 mol/L NaCl solution, flow velocity
It is 1.5BV/h, regulation NaCl solution pH is to 5.0-5.5 for the first time, second of regulation NaCl solution pH to 9.5-10.0；Use again
2BV 1.5 mol/L NaCl solution washing, flow velocity is 0.5BV/h；Elution liquaemin includes：Be 40-50 DEG C with 2BV temperature,
Concentration elutes for 2.5-3.5 mol/L NaCl solution, and flow velocity is 0.5BV/h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710288789.9A CN107056966A (en) | 2017-04-27 | 2017-04-27 | A kind of process for purification of liquaemin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710288789.9A CN107056966A (en) | 2017-04-27 | 2017-04-27 | A kind of process for purification of liquaemin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107056966A true CN107056966A (en) | 2017-08-18 |
Family
ID=59604061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710288789.9A Pending CN107056966A (en) | 2017-04-27 | 2017-04-27 | A kind of process for purification of liquaemin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107056966A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110194812A (en) * | 2019-05-29 | 2019-09-03 | 四川农业大学 | One boar lung source refining crude heparin sodium method |
| CN111909288A (en) * | 2020-08-13 | 2020-11-10 | 山东辰龙药业有限公司 | Refining method of heparin sodium |
| CN111978434A (en) * | 2020-08-26 | 2020-11-24 | 山东辰龙药业有限公司 | Separation and purification method of refined heparin sodium |
| CN113831423A (en) * | 2021-10-20 | 2021-12-24 | 北京赛而生物药业有限公司 | Method for reducing content of dermatan sulfate in heparin sodium |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643369A (en) * | 2011-03-21 | 2012-08-22 | 如皋市坝新肠衣有限公司 | Resin separation and purification process of heparin sodium |
| CN102977228A (en) * | 2012-12-06 | 2013-03-20 | 如皋市坝新肠衣有限公司 | Method for extracting high-potency heparin sodium |
| CN103588902A (en) * | 2013-10-31 | 2014-02-19 | 安徽工贸职业技术学院 | Separation purification method of refined heparin sodium |
| WO2016137471A1 (en) * | 2015-02-26 | 2016-09-01 | Nantpharma, Llc | Method for enhanced heparin quality |
-
2017
- 2017-04-27 CN CN201710288789.9A patent/CN107056966A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643369A (en) * | 2011-03-21 | 2012-08-22 | 如皋市坝新肠衣有限公司 | Resin separation and purification process of heparin sodium |
| CN102977228A (en) * | 2012-12-06 | 2013-03-20 | 如皋市坝新肠衣有限公司 | Method for extracting high-potency heparin sodium |
| CN103588902A (en) * | 2013-10-31 | 2014-02-19 | 安徽工贸职业技术学院 | Separation purification method of refined heparin sodium |
| WO2016137471A1 (en) * | 2015-02-26 | 2016-09-01 | Nantpharma, Llc | Method for enhanced heparin quality |
Non-Patent Citations (1)
| Title |
|---|
| 陈立新等: "《功能塑料（第1版）》", 30 June 2004, 化学工业出版社 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110194812A (en) * | 2019-05-29 | 2019-09-03 | 四川农业大学 | One boar lung source refining crude heparin sodium method |
| CN111909288A (en) * | 2020-08-13 | 2020-11-10 | 山东辰龙药业有限公司 | Refining method of heparin sodium |
| CN111909288B (en) * | 2020-08-13 | 2021-09-03 | 山东辰龙药业有限公司 | Refining method of heparin sodium |
| CN111978434A (en) * | 2020-08-26 | 2020-11-24 | 山东辰龙药业有限公司 | Separation and purification method of refined heparin sodium |
| CN113831423A (en) * | 2021-10-20 | 2021-12-24 | 北京赛而生物药业有限公司 | Method for reducing content of dermatan sulfate in heparin sodium |
| CN113831423B (en) * | 2021-10-20 | 2023-02-03 | 北京赛而生物药业有限公司 | Method for reducing content of dermatan sulfate in heparin sodium |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105294790B (en) | A method of extracting high-purity stevioside from STEVIA REBAUDIANA | |
| CN107056966A (en) | A kind of process for purification of liquaemin | |
| CN110272932B (en) | Preparation method of ganoderma lucidum spore powder polysaccharide peptide | |
| CN101972479A (en) | Preparation process of intravenous injection human immunoglobulin | |
| CN106146278B (en) | A kind of technique for extracting separation Co-Q10 from bacteria residue | |
| CN101613390A (en) | A kind of method of separating and purifying high-purity cordycepin | |
| CN102351917A (en) | Method for extracting raffinose from cotton seed meal | |
| CN105777826A (en) | Method for extracting flavone from dendrocalamus latiflorus leaves | |
| CN106800586A (en) | A kind of method of Moringa protein high efficiency extraction | |
| CN102477104A (en) | Method for separating and purifying polysaccharide from Hovenia acerba | |
| CN106754834A (en) | A kind of preparation technology of high activity papain | |
| CN102503804B (en) | Method for continuously decoloring succinic acid fermentation liquor by using activated carbon | |
| CN110437290A (en) | A kind of steviol glycoside extracting and developing and purification process | |
| CN110627634B (en) | Method for separating and extracting lactic acid from daqu liquor by-product yellow water | |
| CN106045959B (en) | A kind of method that glucosidase procyanidins are prepared using grape seed extract | |
| CN104558227A (en) | Method for extracting ganoderan | |
| CN102584611B (en) | Production method for medical grade valine | |
| CN107098989A (en) | A kind of preparation method of liquaemin | |
| CN111018939A (en) | Rapid refining method of tea saponin | |
| CN103641886B (en) | A kind of process for purification of glutamine dipeptide | |
| CN100509757C (en) | Purification method of *N-L-arginine | |
| CN107721965A (en) | The extraction process of litchi rind OPC | |
| CN107022039A (en) | A kind of new method that liquaemin is prepared by raw material of chitterlings | |
| CN1156289C (en) | Preparation of American ginseng extract (CNT-2000) without pesticide residues | |
| CN102180781B (en) | Method for extracting and producing high-purity xanthohumol from residues generated by extracting hops by carbon dioxide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170818 |