CN104479051A

CN104479051A - Technology for preparing high purity low molecular weight heparin sodium

Info

Publication number: CN104479051A
Application number: CN201410833249.0A
Authority: CN
Inventors: 刘冠男; 尹倩; 夏衬来
Original assignee: QINGDAO JIULONG BIO-PHARMACEUTICAL Co Ltd
Current assignee: QINGDAO JIULONG BIO-PHARMACEUTICAL Co Ltd
Priority date: 2014-12-29
Filing date: 2014-12-29
Publication date: 2015-04-01

Abstract

The invention discloses a technology for preparing high purity low molecular weight heparin sodium and belongs to the field of biological engineering. The technology mainly comprises the steps of salting out for purification, material supplement, heating-up, oxidation, gel elution, ion exchange, ethanol precipitation, vacuum drying and the like. The technology solves the problems that heparin sodium analogues are difficult to remove, the purity cannot meet medicinal requirements, and the preparation technology is complicated and the like at present. The technology realizes preparation and decoloration of low molecular weight heparin sodium by a material supplement, heating-up and oxidation method, weakly-basic ion exchange resin is used for effectively removing the heparin sodium analogues, a production cycle is shortened, the cost is saved, and the technology is simple easy to implement and suitable for industrial production.

Description

A kind of technique preparing high-purity low-molecular heparin sodium

Technical field

What the present invention relates to a kind of biological medicinal raw material prepares purification process, belongs to bioengineering field, specifically a kind of technique preparing high-purity low-molecular heparin sodium.

Background technology

Heparin sodium is a kind of rich anion-containing mucopolysaccharide sulfuric ester, is produced by the mastocyte in animal body and alkaline granulocyte, because extracting from liver at first, and therefore named heparin.The heparin of Clinical practice extracts from animal intestine, lung mucous membrane, vessel wall etc. the sodium salt obtained, and heparin sodium has anticoagulation, anti-inflammatory, the various biological function such as anticancer, but life-time service has the negative interactions such as such as hemorrhage, fracture.Phase early 1980s finds that low molecular sodium heparin has the well less negative interaction of stronger activity, becomes main clinical anticoagulant.

Had more than 10 kind of LMWH to come out one after another abroad at present, the whole world four large pharmacy corporations all have the sale of this series products, and China is main heparin raw material state, and output accounts for the half in the whole world.Being applied to clinical LMWH is be that raw material prepares by unfractionated heparin, and preparation method mainly contains biological enzyme, chemical cleavage method two kinds.They respectively have superiority, and heparinase degradation conditions is gentle, specificity is high, by product is few and introduce a ultraviolet sensual pleasure group, easy to detect, but enzyme is expensive, is difficult to cross over industrial boundary.Chemical cleavage method inevitably changes the structure, impact activity etc. of heparin sodium.The patent (grant number CN096034) of China discloses a kind of heparin fraction its preparation method of purifying and contains their composition.The heparin fraction of purifying is prepared as follows: in heparin sodium, add nitrite, pH0.8-6 process 10-60 minute, again in alkaline media with sodium borohydride reduction, destroy excess sodium borohydrate with HCL and use alcohol settling classification, under the solution obtained is exposed to UV irradiation systems, temperature is 3 DEG C-55 DEG C, and pH is 2-10, chromatogram purification, is finally separated to obtain explanation heparin sodium with alcohol settling with sodium-chlor.United States Patent (USP) (publication number 3099600) discloses a kind of method of chromatographic technique separation and purification heparin sodium: be dissolved in by heparin sodium crude in buffered soln, add ion exchange resin TEAM-cellulose or DEAE-ellulose of 3-15 times of heparin sodium weight, after use buffer solution, eluting salt, alcohol settling, dry product.Aforesaid method is the process of industrial typical preparation and purified heparin sodium, and complex manufacturing, cost are high, and does not mention the minimizing technology of this major impurity of analog chondroitin polysulfate to heparin sodium.

Summary of the invention

Object of the present invention provides a kind of technique preparing high-purity low-molecular heparin sodium to improve the deficiencies in the prior art; This invention provide a kind of easy Low molecular heparin you preparation, purification technique, obtained low molecular sodium heparin molecular-weight average is at about 4.7KDa, tire and be not less than 178U/mg, higher than the 170U/mg that Chinese Pharmacopoeia requires, it is qualified to cut according to the detection of Chinese Pharmacopoeia heparin sodium examination criteria.

Technical scheme of the present invention is: a kind of technique preparing high-purity low-molecular heparin sodium, and its concrete steps are as follows:

(1) be dissolved in Nacl solution by crude heparin sodium, mechanical stirring is all dissolved to heparin sodium, adds alkali lye until produce precipitation in solution, leaves standstill rear mistake completely and filters insolubles, obtain filtrate;

(2) by filtrate in step (1) with lye pH adjustment to 7.5-12.5, add H ₂o ₂solution, reacts 2-5 hours, obtains reaction solution at 5-33 DEG C;

(3) reaction solution that step (2) obtains is warming up to 25-55 DEG C, after this adds H every 2-5 hours ₂o ₂solution, and regulate pH; Coreaction 12-25h after feed supplement;

(4) add sorbent material in the reaction solution obtained to step (3), stir and cross filtering insolubles, collect filtrate;

(5) filtrate step (4) obtained, by the chromatographic column balanced, collects low-molecular-weight heparin sodium elutriant;

(6) the elutriant weak-base ion-exchange resin that step (5) is collected is adsorbed, use the Nacl eluant solution of three kinds of concentration respectively, collect the first Nacl elutriant with the third concentration and mix;

(7) by the mixing elutriant in step (6), be cooled to-8-11 DEG C under magnetic stirring, then obtain product with alcohol settling, vacuum-drying.

In preferred steps (1), the mass percentage concentration of Nacl solution is 1.0-2.1%; The consumption of Nacl solution is 1.8-3.2mL/g heparin sodium crude amount; Time of repose is 0.9-3 hours.The alkali lye of step (1) and step (2) is NaOH or KOH solution; The concentration of alkali lye is 0.5-2.5mol/L.

The H added in preferred steps (2) and step (3) ₂o ₂the volume ratio of solution and Nacl solution is 2-6:100; H in step (2) ₂o ₂the concentration of solution is 0.5-5.5mol/L, H in step (3) ₂o ₂the concentration of solution is 0.6-3.4mol/L.Step has added H in (3) at every turn ₂o ₂measure pH after solution, make pH between 6.4-8.0.

Sorbent material described in preferred steps (4) is gac, bentonite or silicate; The add-on of sorbent material is 2.2-5.5g/100mlNacl amount of solution.

Gel chromatographic columns described in preferred steps (5), sample layer is 10-30% column volume, and the footpath/height of post is 1:3-8; With mass percentage concentration 2-7%Nacl solution equilibria and wash-out, flow velocity is column volume/1.0-2.5 hour, elutriant during mobile phone 40-100 minutes.

In preferred steps (6), the Nacl solution of three kinds of concentration is respectively 0.05-0.45g/L, 0.35-0.65g/L, 0.60-0.95g/L; Its elution time is 1.6-2.9 hours, and sample layer is 10-30% column volume.

beneficial effect:

The present invention adopts feed supplement heating and oxidation coupled ion exchange resin legal system standby and purifying obtains molecular-weight average 4.7KDa low molecular sodium heparin, and it is tired and is not less than 178U/mg, and the rate of recovery is not less than 75%.(2) add H2O2 by intensification and can effectively control heparin sodium Oxidative inactivation, and achieve oxidation with decolouring and synchronously carry out.(3) difference of negative ion intensities contained by heparin sodium and analog chondroitin polysulfate thereof, weak base anion-exchange resin is adopted to adsorb all chondroitin polysulfates and a small amount of heparin sodium, finally use low concentration of salt stepwise elution, obtain heparin sodium product.The weak base anion-exchange resin that test adopts, chondroitin polysulfate impurity is adsorbed, and heparin sodium product directly flows out, and with a small amount of heparin sodium that substep salt method wash-out adsorbs, greatly improves its rate of recovery.The low molecular sodium heparin product finally obtained reaches Chinese Pharmacopoeia standard, and after testing not containing chondroitin polysulfate composition.

Embodiment

Explain the present invention further below in conjunction with example, but case study on implementation does not do any type of restriction to the present invention.

Be dissolved in 150mL mass percentage concentration 1.6%NaCI solution by 50 grams of crude heparin sodiums, mechanical stirring is all dissolved to heparin sodium, adds 1.2mol/LKOH until produce precipitation, leaves standstill 1.5 hours, cross to filter and do not allow thing, obtain filtrate in solution; Adjust pH to 10.5 with 1.2mol/LKOH, add the H of 7.5mL 5moi/L ₂o ₂, 30 DEG C, react 3.5 hours.Reaction solution is warming up to 50 DEG C DEG C, after this adds the H of 7.5mL 3mol/L every 3 hours ₂o ₂, maintain pH7.5, after feed supplement, coreaction 15h. adds 4.5g bentonite in reaction solution, magnetic agitation, crosses filtering and does not allow thing, obtain filtrate; With mass percent 6%NaCL solution equilibria Sephadex G-25 post, sample layer is 25% column volume, and post footpath/height is 1:7, by filtrate by this post, with mass percent 6%NaCL wash-out, flow velocity is column volume/1.8 hour, collects 7.-90min elutriant; Eluent stream through 330 chromatographic columns, sample layer is 25% column volume, and plant height/footpath is 8:1, respectively with 0.3 .6,0.9g/LNaCL wash-out 2.5h, collect 0.3,0.9g/L elutriant; By this elutriant, be cooled to 8 DEG C under magnetic stirring, after this obtain product with alcohol settling, vacuum-drying.Footpath is detected, and it tires as 187U/mg, the rate of recovery 76.8%, chondroitin sulfate 0004mg/mL, dermatan sulfate 0.003mg/mL, and indices all meets Chinese Pharmacopoeia requirement.

Claims

1. prepare a technique for high-purity low-molecular heparin sodium, its concrete steps are as follows:

(1) be dissolved in Nacl solution by crude heparin sodium, mechanical stirring is all dissolved to heparin sodium, adds alkali lye until produce precipitation, leaves standstill, cross and filter insolubles, obtain filtrate in solution;

(2) by filtrate in step (1) with lye pH adjustment to 7.5-12.5, add H2O2 solution, react 2-5 hours at 5-33 DEG C, obtain reaction solution;

(3) reaction solution that step (2) obtains is warming up to 25-55 DEG C, after this added H2O2 solution every 2-5 hours, and regulate pH; Coreaction 12-25h after feed supplement;

(4) add sorbent material in the reaction solution obtained to step (3), stir and filter, obtaining filtrate;

(6) the elutriant weak-base ion-exchange resin collected in step (5) is adsorbed, use the Nacl eluant solution of three kinds of concentration respectively, collect the first Nacl elutriant with the third concentration and mix;

(7) the mixing elutriant will collected in step (6), is cooled to-8-11 DEG C under magnetic stirring, then obtains product with alcohol settling, vacuum-drying.

2. technique according to claim 1, is characterized in that: in step (1), the mass percentage concentration of Nacl solution is 1.0-2.1%; The consumption of Nacl solution is 1.8-3.2mL/g heparin sodium crude amount; Time of repose is 0.9-3 hours.

3. technique according to claim 1, is characterized in that: the alkali lye of step (1) and step (2) is NaOH or KOH solution; The concentration of alkali lye is 0.5-2.5mol/L.

4. technique according to claim 1, is characterized in that: the H2O2 solution added in step (2) and step (3) and the volume ratio of Nacl solution are 2-6:100; In step (2), the concentration of H2O2 solution is 0.5-5.5mol/L, and in step (3), the concentration of H2O2 solution is 0.6-3.4mol/L.

5. technique according to claim 1, is characterized in that: measure pH after having added H2O2 solution in step (3) at every turn, make pH between 6.4-8.0.

6. technique according to claim 1, is characterized in that: the sorbent material described in step (4) is gac, bentonite or silicate; The add-on of sorbent material is 2.2-5.5g/100mlNacl amount of solution.

7. technique according to claim 1, is characterized in that: the gel chromatographic columns described in step (5) is Sephadex, and sample layer is 10-30% column volume, and the footpath/height of post is 1:3-8; With mass percentage concentration 2-7%Nacl solution equilibria and wash-out, flow velocity is column volume/1.0-2.5 hour, elutriant during mobile phone 40-100 minutes.

8. technique according to claim 1, is characterized in that: in step (6), the Nacl solution of three kinds of concentration is respectively 0.05-0.45g/L, 0.35-0.65g/L, 0.60-0.95g/L; Its elution time is 1.6-2.9 hours, and sample layer is 10-30% column volume.