CN104558251B - A kind of preparation method of liquaemin - Google Patents
A kind of preparation method of liquaemin Download PDFInfo
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- CN104558251B CN104558251B CN201510055870.3A CN201510055870A CN104558251B CN 104558251 B CN104558251 B CN 104558251B CN 201510055870 A CN201510055870 A CN 201510055870A CN 104558251 B CN104558251 B CN 104558251B
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- liquaemin
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Abstract
The invention discloses a kind of liquaemin novel preparation method.The method is pre-processed with heparin sodium crude as raw material using salt solution technique, then oxidized, ion exchange resin absorption, washing, wash-out, most afterwards through ultrafiltration, lyophilized acquisition liquaemin.The invention has the advantages that with short production cycle, production is consumed energy, low, product quality is high, does not use organic solvent, is conducive to environmental protection, is suitable for large-scale industrial production.
Description
Technical field
The invention belongs to biochemical drug production technical field, and in particular to a kind of preparation method of liquaemin.
Background technology
Heparin is gained the name because being extracted from the liver of animal earliest, is a kind of natural anticoagulative substance.Heparin quilt in 1918
Chance on obvious anticoagulation, nineteen thirty-five starts to be used for clinic, is incorporated into American Pharmacopeia the forties in 20th century
Used as a kind of clinical anticoagulation medicine, existing more than 70 year, is the maximum anticoagulant of most effective in the world and quantity so far
Thing, because heparin molecule textural anomaly is complicated, molecular weight is difficult to fix, in a short time cannot artificial chemistry synthesis, only come at present
The heparin for coming from pig intestinal mucosa can be used in clinical treatment.Exactly because liquaemin is natural by what is extracted in organism
Material, is not the product of chemical synthesis, is favored by consumers in general in Clinical practice, therefore also just as in the world ten
Divide the biochemical drug for selling well.
It is more to the preparation method of liquaemin both at home and abroad at present, such as using repeatedly oxidation, the method [Shaanxi of multiple alcohol precipitation
University of Science and Technology's journal, 2005,23 (5)：32-34] or ion exchange, oxidation, method [amino acid and the biological money of multiple alcohol precipitation
Source, 2000,22 (2)：19~21] refined.But these techniques are present, and the production cycle is long, titer of heparin sodium is low or organic molten
The shortcomings of agent consumption is big.Therefore new liquaemin preparation technology is developed, effectively improves product quality, reducing energy consumption, reduction environmental protection
Pressure is still the problem that those skilled in the art need to solve.
The content of the invention
It is an object of the present invention to overcome the above deficiencies, there is provided a kind of with short production cycle, production low, product matter of power consumption
Amount is high, environmentally friendly preparation method.The present invention is using the step such as salt solution, purifying resin simultaneous oxidation decolouring, ultrafiltration, lyophilized
High-quality liquaemin is obtained.This is simple for process, is suitable to industrial-scale production.
The inventive method the following specifically describes below：
A kind of preparation method of liquaemin, the method is comprised the following steps：
1) salt solution：Sodium chloride is added after heparin sodium crude purifying water dissolves, while adjusting pH value carries out salt to 8.5-9.0
Solution, then through centrifugation, obtain liquaemin pretreatment fluid；
2) ion exchange：Liquaemin pretreatment fluid is down to 28-32 DEG C, hydrogen peroxide is added, then add to strong base it is cloudy from
Adsorbed in sub-exchange resin post, washed, eluted, collect liquaemin eluent；
3) ultrafiltration：After the liquaemin eluent regulation pH value of collection to 5.5~6.0 ultrafiltrate will be obtained through ultrafiltration；
4) freeze：Ultrafiltrate is crossed into 0.22um filter membranes, gained filtrate is freeze-dried to obtain liquaemin.
Wherein step 1) described in purified water, it is 8~10ml/g that purified water adds the ratio of volume and heparin sodium crude weight；
The ratio of the sodium chloride addition and purifying water consumption volume is 1/100~2/100g/ml；Adjust the hydrogen of pH value any concentration
Sodium hydroxide solution.
Wherein step 1) in salt solution temperature 50 C~60 DEG C, the salt solution time is 2~3 hours.
Wherein step 2) in hydrogen peroxide to add volume be the 1%~2% of liquaemin pretreatment fluid volume.
Wherein step 2) in, strong basic type anion-exchange resin is OC1074 or FPA-98.
Wherein step 2) in, ion exchange absorption speed 0.05~0.06 times of resin column volume (0.05- per hour
0.06BV/h)。
Wherein step 2) in, washing solution used is 5.5%~6% sodium chloride solution, 0.5 times per hour of washing speed
Resin column volume (0.5BV/h).5.5%~6% sodium chloride solution adds water constant volume to 100ml for the sodium chloride of 5.5g~6g,
It is as follows.
Wherein step 2) in, wash-out solution used is 12% sodium chloride solution, elution speed per hour 0.05~0.06
Times resin column volume (0.05~0.06BV/h).
Wherein step 3) in regulation pH value any concentration hydrochloric acid solution.
Wherein step 3) in the milipore filter that uses of ultrafiltration be the film of molecular cut off 1000.
The device have the advantages that：
1st, whole preparation technology is more succinct, and ion-exchange step completes oxidative decoloration, isolating protein, nucleic acid and class simultaneously
Heparin, cycle is short consumes energy low, with important economic worth.
2nd, organic solvent is not used in preparation technology, realizes green production.
3rd, the heparin sodium product quality for preparing is high.
In the present invention, hydrogen peroxide is added into liquaemin pretreatment fluid, oxidative decoloration process and resin before upper resin column
Absorption, purge process are carried out simultaneously, have initiated the technique that oxidation solution directly goes up resin column.The technique saves what is individually aoxidized
Time；And the classification wash-out after finishing is adsorbed by resin column, effectively go isolating protein, nucleic acid and heparan, and a step to complete
Removal of impurities is decolourized, and can remove multiple alcohol precipitation and re-oxidation step from, reduces energy consumption, and the production cycle at least saves the time 24 hours, it is to avoid
The use of organic solvent, is conducive to environmental protection；The heparin sodium product quality for preparing is high, and potency is higher than 180U/mg, A260nm
Absorbance is less than 0.1, and protein content is less than 0.5%, and European Pharmacopoeia regulation is met about material and molecular weight.
Specific embodiment
Following embodiments are used merely to explain realizes the method for the present invention, should not be construed as limiting the invention, Suo Youji
The modifications and variations made in thinking of the invention all should be attributed to protection scope of the present invention.
Heparin sodium crude (potency is 78U/mg) used in the present invention is that the female Hebei biotechnology of North China pharmacy China is limited
Company is made；Hydrogen peroxide (commercially available, 30% content), NaOH, sodium chloride are commercially available；Ion exchange resin OC1074 is Germany
Lanxess Corporation's product, FPA-98 are U.S.'s ROHM AND HAAS product.
Embodiment 1
1 kilogram of taking heparin sodium crude product, water dissolves are purified with 8L, add 80g sodium chloride, rise high-temperature to 50 DEG C, while with
3mol/L sodium hydroxide solutions regulation pH value is 8.5, with this understanding salt solution 3 hours, and salt solution liquid is centrifuged to obtain liquaemin pretreatment
Liquid.Liquaemin pretreatment fluid temperature is down to 28 DEG C, hydrogen peroxide is added, its volume is the 1% of liquaemin pretreatment fluid volume, is stirred
Mix it is uniform after add to and adsorbed in the resin column equipped with 15L OC1074 resins, control adsorption rate for 0.05BV/h, adsorb
Resin after finishing first is washed with 5.5% sodium chloride solution, flow velocity 0.5BV/h, washing to absorbance A260nm≤0.20、A280nm
When≤0.20, eluted with 12% sodium chloride solution, elution speed 0.05BV/h, detect lower column liquid refractive power, when refractive power is more than 5.5
Start to collect eluent, about collect 1BV.It is 5.5 that the eluent of collection 3mol/L hydrochloric acid solutions adjust pH, then through 1000 molecules
The milipore filter of amount, gained ultrafiltrate freezes to obtain liquaemin finished product 1, and titer yield 86.5%, its testing result is shown in Table 1.
Embodiment 2
1 kilogram of taking heparin sodium crude product, water dissolves are purified with 10L, add 200g sodium chloride, rise high-temperature to 60 DEG C, while
It is 9.0 to adjust pH value with 2mol/L sodium hydroxide solutions, salt solution 2 hours with this understanding, salt solution liquid be centrifuged liquaemin is located in advance
Reason liquid.Liquaemin pretreatment fluid temperature is down to 32 DEG C, hydrogen peroxide is added, its volume is the 2% of liquaemin pretreatment fluid volume,
Added to after stirring and adsorbed in the resin column equipped with 15L FPA98 resins, control adsorption rate for 0.06BV/h, absorption
Resin after finishing first is washed with 6% sodium chloride solution, flow velocity 0.5BV/h, washing to absorbance A260nm≤0.20、A280nm≤
When 0.20, eluted with 12% sodium chloride solution, elution speed 0.06BV/h, detect lower column liquid refractive power, started when refractive power is more than 6
Eluent is collected, 1BV is about collected.It is 6.0 that the eluent of collection 1mol/L hydrochloric acid solutions adjust pH, then through 1000 molecular weight
Milipore filter, gained ultrafiltrate freezes to obtain liquaemin finished product 2, and titer yield 83.0%, its testing result is shown in Table 1.
Embodiment 3
1 kilogram of taking heparin sodium crude product, water dissolves are purified with 10L, add 200g sodium chloride, rise high-temperature to 50 DEG C, while
It is 8.5 to adjust pH value with 1mol/L sodium hydroxide solutions, salt solution 2 hours with this understanding, salt solution liquid be centrifuged liquaemin is located in advance
Reason liquid.Liquaemin pretreatment fluid temperature is down to 30 DEG C, hydrogen peroxide is added, its volume is liquaemin pretreatment fluid volume
1.5%, added to after stirring and adsorbed in the resin column equipped with 15L FPA98 resins, it is 0.05BV/ to control adsorption rate
H, the resin after absorption is finished first is washed with 5.5% sodium chloride solution, flow velocity 0.5BV/h, washing to absorbance A260nm≤0.20、
A280nmWhen≤0.20, eluted with 12% sodium chloride solution, elution speed 0.05BV/h, lower column liquid refractive power is detected, when refractive power is more than
Start to collect eluent when 5.5, about collect 1BV.It is 5.5 that the eluent of collection 8mol/L hydrochloric acid solutions adjust pH, then is passed through
The milipore filter of 1000 molecular weight, gained ultrafiltrate freezes to obtain liquaemin finished product 3, and titer yield 86.9%, its testing result is shown in Table
1。
Embodiment 4
1 kilogram of taking heparin sodium crude product, water dissolves are purified with 9L, add 135g sodium chloride, rise high-temperature to 55 DEG C, while with
4mol/L sodium hydroxide solutions regulation pH value is 9.0, salt solution 2.5 hours with this understanding, salt solution liquid be centrifuged liquaemin is located in advance
Reason liquid.Liquaemin pretreatment fluid temperature is down to 30 DEG C, hydrogen peroxide is added, its volume is the 1% of liquaemin pretreatment fluid volume,
Added to after stirring and adsorbed in the resin column equipped with 15L OC1074 resins, control adsorption rate for 0.06BV/h, inhaled
It is attached finish after resin first washed with 5.5% sodium chloride solution, flow velocity 0.5BV/h, washing is to absorbance A260nm≤0.20、
A280nmWhen≤0.20, eluted with 12% sodium chloride solution, elution speed 0.06BV/h, lower column liquid refractive power is detected, when refractive power is more than
Start to collect eluent when 5.5, about collect 1BV.It is 5.8 that the eluent of collection 4mol/L hydrochloric acid solutions adjust pH, then is passed through
The milipore filter of 1000 molecular weight, gained ultrafiltrate freezes to obtain liquaemin finished product 4, and titer yield 85.9%, its testing result is shown in Table
1。
The testing result of the embodiment 1-4 of table 1 gained liquaemin finished products
Claims (4)
1. a kind of preparation method of liquaemin, it is characterised in that the method comprises the steps：
1) salt solution：Sodium chloride is added after heparin sodium crude purifying water dissolves, while adjusting pH value carries out salt solution to 8.5~9.0,
Again through centrifugation, liquaemin pretreatment fluid is obtained；
2) ion exchange：Liquaemin pretreatment fluid temperature is down to 28~32 DEG C, hydrogen peroxide is added, then add to strong base it is cloudy from
Adsorbed in sub-exchange resin post, washed, eluted, collect liquaemin eluent, wherein, hydrogen peroxide directly goes up resin column, makes oxidation
Decolorization and resin adsorption, purge process are carried out simultaneously, and a step completes removal of impurities and decolourizes；
3) ultrafiltration：After the liquaemin eluent regulation pH value of collection to 5.5~6.0 ultrafiltrate will be obtained through ultrafiltration；
4) freeze：Ultrafiltrate is crossed into 0.22um filter membranes, gained filtrate is freeze-dried to obtain liquaemin；
Wherein, step 1) described in purified water, it is 8~10ml/g that purified water adds the ratio of volume and heparin sodium crude weight；Institute
The ratio for stating sodium chloride addition and purifying water consumption volume is 1/100~2/100g/ml；Step 1) in salt solution temperature be 50 DEG C~
60 DEG C, the salt solution time is 2~3 hours；Step 2) in add hydrogen peroxide volume for liquaemin pretreatment fluid volume 1%-
2%；Step 2) in, strong basic type anion-exchange resin is OC1074 or FPA-98；Step 2) in, the speed of ion exchange absorption
For 0.05~0.06 times of resin column volume per hour；Washing solution used is 5.5%~6% sodium chloride solution, washs speed per hour
Spend for 0.5 times of resin column volume per hour；Step 2) in, wash-out solution used is 12% sodium chloride solution, speed during wash-out
For 0.05~0.06 times of resin column volume per hour；
Wherein, step 2) in, washing to absorbance A260nm≤0.20、A280nmWhen≤0.20, eluted with 12% sodium chloride solution；Inspection
Lower column liquid refractive power is surveyed, starts to collect eluent when refractive power is more than 5.5.
2. method according to claim 1, wherein step 1) in regulation pH value sodium hydroxide solution.
3. method according to claim 1, wherein step 3) in regulation pH value hydrochloric acid solution.
4. method according to claim 1, wherein step 3) in the milipore filter that uses of ultrafiltration be molecular cut off 1000
Film.
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| CN104817651A (en) * | 2015-05-26 | 2015-08-05 | 苏州鸿洋医药科技有限公司 | Refinement technique of heparin sodium |
| CN111939121B (en) * | 2020-07-13 | 2022-03-08 | 华北制药华坤河北生物技术有限公司 | Heparin sodium tube-sealing injection and preparation method thereof |
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| US3179566A (en) * | 1963-05-16 | 1965-04-20 | Canada Packers Ltd | Purification of heparin |
| CN1844165A (en) * | 2006-03-22 | 2006-10-11 | 南京健友生物化学制药有限公司 | Process for preparing high purity sodium heparin by purification of crude sodium heparin |
| 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 |
| CN103214597B (en) * | 2013-05-14 | 2015-11-11 | 枣庄赛诺康生化股份有限公司 | Decoloration method for enoxaparin sodium intermediate |
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