CN108610436A - A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate - Google Patents
A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate Download PDFInfo
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- CN108610436A CN108610436A CN201810265492.5A CN201810265492A CN108610436A CN 108610436 A CN108610436 A CN 108610436A CN 201810265492 A CN201810265492 A CN 201810265492A CN 108610436 A CN108610436 A CN 108610436A
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- 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
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J47/00—Ion-exchange processes in general; Apparatus therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J49/00—Regeneration or reactivation of ion-exchangers; Apparatus therefor
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- 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
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
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Abstract
The present invention relates to a kind of methods of separation of glasses acid sodium and heterozygosis chondroitin sulfate, include the following steps：(1) loading：By sodium hyaluronate dissolution of raw material in water, sample solution is obtained；It will be in sample solution loading to Q Sepharose fast flow anion-exchange columns；(2) gradient elution：Gradient elution is carried out with the NaCl aqueous solutions of 0.4M, 0.6M and 1.0M successively, the eluent of 0.4M NaCl aqueous solutions is collected, for the solution containing sodium hyaluronate；The eluent for collecting 1.0M NaCl aqueous solutions, for the solution containing heterozygosis chondroitin sulfate.The present invention realizes the purpose for disposably removing multiple heterozygosis chondroitin sulfate impurity and being efficiently separated with sodium hyaluronate, it is separated go out heterozygosis chondroitin sulfate purity reach 99.5wt% or more, while sodium hyaluronate obtains efficiently purifying, and purity reaches 99.5wt% or more.
Description
Technical field
The present invention relates to technical field of bioengineering more particularly to a kind of separation of glasses acid sodium and heterozygosis chondroitin sulfate
Method.
Background technology
Sodium hyaluronate is a kind of macromolecule polysaccharide, for surgery arthropathy, tissue repair, ophthalmologic operation, auxiliary diagnosis etc.
Aspect.Its production technology is broadly divided into two major classes, using animal tissue as the extraction method of raw material and bacterial fermentation process.It is extracted in cockscomb
It is controllable best by the measurement to sodium hyaluronate purity during method (animal tissue) produces the technical study of sodium hyaluronate
Process conditions, provide more accurate data for process modification and support, to ensure the quality of downstream takeaway.
CN102516411A discloses a kind of pre-treating method of sodium hyaluronate fermentation liquid, after kieselguhr adsorption, passes through object
The filtering of reason method and micro-filtration reach the degerming purpose of sodium hyaluronate fermentation liquid, at low cost, safely, effectively；Utilize we
The sodium hyaluronate fermentation liquid of method processing, can remove thalline in zymotic fluid, effectively reduce containing for the impurity such as nucleic acid and albumen substantially
Amount is particularly suitable for the industrialized production of pharmaceutical grade sodium hyaluronate, and by the saturating of the processed sodium hyaluronate fermentation liquid of this method
Light rate is relatively high, but this method does not mention the heterozygosis chondroitin sulfate isolated in sodium hyaluronate, and cannot get pure
Spend higher heterozygosis chondroitin sulfate.
CN206392032U discloses a kind of purification devices of cross-linking sodium hyaluronate gel, and ion is arranged in autoclave body and hands over
Column processing structure is changed, including strong-acid type cation exchange column and strong base anion exchange column, mainly ion is utilized to hand over
Changing column goes this amphoteric impurity of removing protein, protein denaturation to be unable to get the heterozygosis chondroitin sulfate that can be recycled.
Injection and cornea preserve in the prior art contains sodium hyaluronate and heterozygosis chondroitin sulfate simultaneously mostly in liquid
Both components.It is generally acknowledged that the heterozygosis chondroitin sulfate contained in sodium hyaluronate is helpful, such as chondroitin sulfate is to cornea
Collagenous fibres have protective effect, can promote the growth of fiber in matrix, enhance permeability, improve blood circulation, accelerate new old
Metabolism, promotes the absorption of penetrating fluid and the elimination of inflammation, but its adverse reaction for being generated in certain clinical application fields, some
Patient have itch, allergic phenomenas, these adverse reactions such as redness are ignored.It is miscellaneous that those skilled in the art do not have motivation to remove this
Matter.And the non-one-component of heterozygosis chondroitin sulfate is constituted, and is not reported in the prior art multiple miscellaneous by a step removal
Matter and the method efficiently separated with sodium hyaluronate.
Invention content
In view of problems of the prior art, one of the objects of the present invention is to provide miscellaneous in a kind of separation of glasses acid sodium
The method of matter efficiently separates out the heterozygosis chondroitin sulfate of high-purity and the sodium hyaluronate of high-purity from sodium hyaluronate raw material.
For this purpose, the present invention adopts the following technical scheme that：
The present invention provides a kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate, includes the following steps：
(1) loading：By sodium hyaluronate dissolution of raw material in water, sample solution is obtained；By sample solution loading to Q-
In Sepharose fast flow anion-exchange columns；
(2) gradient elution：Gradient elution is carried out with the NaCl aqueous solutions of 0.4M, 0.6M and 1.0M successively, collects 0.4M
The eluent of NaCl aqueous solutions, for the solution containing sodium hyaluronate；The eluent of 1.0M NaCl aqueous solutions is collected, it is miscellaneous to contain
Close the solution of chondroitin sulfate.
" comprising " of the present invention, it is intended that it can also include other steps, these other steps in addition to the step
Assign the method different effects.In addition to this, " comprising " of the present invention may be replaced by enclosed " for ".
The non-one-component of heterozygosis chondroitin sulfate is constituted, and the multiple heterozygosis of disposable removal were not reported still in the prior art
Chondroitin sulfate impurity and the method efficiently separated with sodium hyaluronate.The method of the invention uses Q-Sepharose fast
Flow anion-exchange columns coordinate the gradient elution of NaCl aqueous solutions, realize that disposably to remove multiple heterozygosis chondroitin sulfates miscellaneous
Matter and the purpose efficiently separated with sodium hyaluronate, it is separated go out heterozygosis chondroitin sulfate purity reach 99.5wt% or more, simultaneously
Sodium hyaluronate obtains efficiently purifying, and purity reaches 99.5wt% or more.
Preferably, step (1) the sodium hyaluronate raw material includes the sodium hyaluronate raw material of biological extraction method production, such as from
With the sodium hyaluronate raw material etc. of biological extraction method production in cockscomb.
Preferably, in step (1) described sample solution sodium hyaluronate raw material a concentration of 0.3~0.8g/L, such as 0.3g/
L, 0.35g/L, 0.4g/L, 0.45g/L, 0.5g/L, 0.55g/L, 0.6g/L, 0.65g/L, 0.7g/L, 0.75g/L or 0.8g/L
Deng preferably 0.4~0.6g/L.
Preferably, the ratio of height to diameter of step (1) the Q-Sepharose fast flow anion-exchange columns be (10~
20):1, such as 10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1 or 20:1 etc., preferably (10~
15):1。
Preferably, flow velocity when step (1) described loading is 5.0~10.0mL/min, such as 5.0mL/min, 5.5mL/
min、6.0mL/min、6.5mL/min、7.0mL/min、7.5mL/min、7.8mL/min、8.0mL/min、8.2mL/min、
8.5mL/min, 9mL/min, 9.5mL/min or 10.0mL/min etc., preferably 7.5~8.5mL/min；
Preferably, loading volume is 0.1~0.3 times of column volume in the step (1), for example, 0.1 times, 0.15 times, 0.2
Again, 0.25 times or 0.3 times etc. so that sample fully, is well adsorbed with the filled media in exchange column, improves separative efficiency.
Preferably, further include before the step (1)：Activate Q-Sepharose fast flow anion-exchange columns.
Preferably, the activation specifically includes：Ultra-pure water, NaCl aqueous solutions and ultra-pure water, the excessively described Q- are used successively
Sepharose fast flow anion-exchange columns are washed, and exchange column is balanced, and are washed away and may wherein be mixed
Extra impurity in being detached to the later stage is further ensured that the purity of gained separation phase.
Preferably, a concentration of 0.2~0.6M of the NaCl aqueous solutions when activation, for example, 0.2M, 0.3M, 0.4M,
0.5M or 0.6M etc..
The present invention is coordinated by the way that sample concentration, elution volume, flow velocity is arranged with eluate concentration gradient, is cooperateed with mutually, into
One step improves the purity of gained sodium hyaluronate and chondroitin sulfate.Preferably, the step (2) elution volume is 10~13
Column volume again, such as 10 times, 10.5 times, 11 times, 11.5 times, 12 times, 12.5 times or 13 times etc..
Preferably, flow velocity when step (2) described gradient elution be 5.0~10.0mL/min, such as 5.0mL/min,
5.5mL/min、6.0mL/min、6.5mL/min、7.0mL/min、7.5mL/min、7.8mL/min、8.0mL/min、8.2mL/
Min, 8.5mL/min, 9mL/min, 9.5mL/min or 10.0mL/min etc., preferably 7.5~8.5mL/min.
Preferably, gradient elution is carried out with the NaCl aqueous solutions of 0M, 0.4M, 0.6M and 1.0M successively in the step (2).
Increase 0M NaCl aqueous solutions first carry out elution also act as will be unadsorbed on the effect that first elutes of compound, further carry
Concentration of the high score from phase.
The NaCl aqueous solutions of 0.6M make 0.4M be transitioned into 1.0M when first separation, ensure the relatively high score detached for the first time from effect
Rate, but its eluent is still mixture, can be collected separately and continue to detach.Preferably, step (2) further includes：It collects
The eluent of 0.6M NaCl aqueous solutions, return to step (1) are detached again.
Preferably, the method further includes step (3)：Solution containing sodium hyaluronate obtained by step (2) is purified,
Obtain sodium hyaluronate；And/or purify the solution containing heterozygosis chondroitin sulfate obtained by step (2), obtain chondroitin sulfate
Element.The sodium hyaluronate that the method for the invention is isolated has high-purity, can be used for producing preparation, treatment knee osteoarthritis, shoulder
The diseases such as all inflammation, while can also be used as the basic substance of standard items.The chondroitin sulfate that the method for the invention obtains also has
Important practical value, such as may be used as treatment neuralgia, nervous migraine, arthralgia, arthritis and omoplate joint
Bitterly, abdominal postoperative pain etc..
As currently preferred technical solution, the separation of glasses acid sodium and the method for heterozygosis chondroitin sulfate include
Following steps：
(1) it activates：The NaCl aqueous solutions and ultra-pure water of ultra-pure water, 0.2~0.6M, the excessively described Q-Sepharose are used successively
Fast flow anion-exchange columns are washed；
Loading：By the sample solution of a concentration of 0.3~0.8g/L with the flow velocity loading of 5.0~10.0mL/min to Q-
In Sepharose fast flow anion-exchange columns, loading volume is 0.1~0.3 times of column volume；
(2) gradient elution：M, 0.4M, 0.6M and 1.0M NaCl aqueous solutions carry out gradient elution, flow velocity be 5.0~
10.0mL/min, the column volume that elution volume is 10~13 times collect the eluent of 0.4M NaCl aqueous solutions, to contain glass
The solution of sour sodium；The eluent for collecting 1.0M NaCl aqueous solutions, for the solution containing heterozygosis chondroitin sulfate；Collect 0.6M
The eluent of NaCl aqueous solutions, return to step (1) are detached again.
(3) it purifies：Solution containing sodium hyaluronate obtained by step (2) is purified, sodium hyaluronate is obtained；And/or it will
The solution containing heterozygosis chondroitin sulfate is purified obtained by step (2), obtains chondroitin sulfate.
Compared with prior art, the present invention at least has the advantages that：
1, the present invention has very good effect to impurity removal in sodium hyaluronate, and reliable branch is provided to improve sodium hyaluronate purity
It holds；
2, the present invention is coordinated by the way that sample concentration, elution volume, flow velocity is arranged with eluate concentration gradient, is cooperateed with mutually,
Further increase gained sodium hyaluronate and chondroitin sulfate purity, it is separated go out heterozygosis chondroitin sulfate purity reach
99.5wt% or more, while sodium hyaluronate obtains efficiently purifying, purity reaches 99.5wt% or more, has important practical value.
Description of the drawings
Fig. 1 is the high-efficient gel filtration chromatography figure of sodium hyaluronate raw material in the embodiment of the present invention 1；
Fig. 2 is the high-efficient gel filtration chromatography figure of 0.4M NaCl eluents in the embodiment of the present invention 1；
Fig. 3 is the high-efficient gel filtration chromatography figure of 0.6M NaCl eluents in the embodiment of the present invention 1；
Fig. 4 is the high-efficient gel filtration chromatography figure of 1.0M NaCl eluents in the embodiment of the present invention 1.
Specific implementation mode
Technical solution to further illustrate the present invention below with reference to the accompanying drawings and specific embodiments.But following reality
The simple example that example is only the present invention is applied, the scope of the present invention, protection model of the invention are not represented or limit
It encloses and is subject to claims.
Embodiment 1
A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate, steps are as follows：
(1) it activates：The NaCl aqueous solutions and ultra-pure water for using ultra-pure water, 0.2M successively, cross Q-Sepharose fast flow
Anion-exchange column is washed；
Loading：The sodium hyaluronate raw material 50mg of the biology extraction from cockscomb is dissolved in 100mL water and obtains sample solution, by sample
Product solution is with the flow velocity loading of 8.0mL/min to ratio of height to diameter for 15:1 Q-Sepharose fast flow anion-exchange columns
In, loading volume is 0.2 times of column volume；
(2) gradient elution：Gradient elution, flow velocity 8.0mL/ are carried out with the NaCl aqueous solutions of 0M, 0.4M, 0.6M and 1.0M
Min, the column volume that elution volume is 10 times, develops the color to the eluent of various concentration NaCl with borax sulfate-carbazole,
The NaCl eluents presentation aubergine of wherein 0.4,0.6,1.0mol/L, show to contain uronic acid in above-mentioned elution solution.Then
Respectively by 0.45 μm of the membrane filtration of NaCl eluents of 0.4,0.6,1.0mol/L, it is detected with high performance liquid chromatography.
Testing result is respectively shown in Fig. 2, Fig. 3 and Fig. 4, from the NaCl eluents for understanding 0.4mol/L in gel filtration chromatography figure only
Contain sodium hyaluronate；Not only contain sodium hyaluronate in the NaCl eluents of 0.6mol/L, but also contains impurity；And the NaCl of 1.0mol/L
Impurity is contained only in eluent.So the eluent of the 0.4M NaCl aqueous solutions of collection is the solution containing sodium hyaluronate；It receives
The eluent of the 1.0M NaCl aqueous solutions of collection is the solution containing heterozygosis chondroitin sulfate, can be with although loss partial impurities
The heterozygosis chondroitin sulfate sample that purity is 99.8wt% and the sodium hyaluronate that purity is 99.9wt% are isolated in guarantee, are realized high
Effect separation.
Wherein, in order to verify the ingredient in raw material, the 1mg sodium hyaluronate base glass acid sodium raw materials medicines of the present embodiment are weighed
It is dissolved in 1mL mobile phases, is configured to the solution of 0.5mg/mL.Divided with high performance gel filtration chromatography after solution is filtered
Analysis, the results are shown in Figure 1：Occur two peaks in gel filtration chromatography figure, the former is sodium hyaluronate, and the latter is the miscellaneous of bulk pharmaceutical chemicals
Matter heterozygosis chondroitin sulfate.
Color developing detection method：The eluent 1mL under each concentration is taken, is respectively placed in tool plug test tube, mixing, it is cold in ice bath
But, and under continuous shaking be slowly added dropwise 0.025mol/L borax sulfuric acid solution 5.0mL, close plug, boiling water bath heat 10min (in
Between shake it is primary), it is rapid cooling, add the ethanol solution 0.2mL of 0.125% carbazole, shake up, 15min is heated in boiling water bath
(centre shaking is primary), is cooled to room temperature.
Method for detecting purity：Liquid-phase condition：Using Agilent1100 high performance liquid chromatographs, chromatographic column is
ShodexOHpakSB-806HQ gel chromatographic columns, 35 DEG C of column temperature, sample size are 20 μ L, mobile phase 0.2mol/LNaCl solution；Inspection
Survey device：Differential refraction detector (RID), 40 DEG C of detector temperature.
Embodiment 2
A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate, steps are as follows：
(1) loading：The sodium hyaluronate raw material 30mg of the biology extraction from cockscomb is dissolved in 100mL water and obtains sample solution,
By sample solution with the flow velocity loading of 5.0mL/min to ratio of height to diameter for 20:1 Q-Sepharose fast flow anion are handed over
It changes in column, the column volume that loading volume is 0.1 times；
(2) gradient elution：Gradient elution, flow velocity 5.0mL/ are carried out with the NaCl aqueous solutions of 0.4M, 0.6M and 1.0M
Min, the column volume that elution volume is 11 times collect the eluent of 0.4M NaCl aqueous solutions, for the solution containing sodium hyaluronate；
The eluent for collecting 1.0M NaCl aqueous solutions realizes separation for the solution containing heterozygosis chondroitin sulfate.
Embodiment 3
A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate, steps are as follows：
(1) it activates：The NaCl aqueous solutions and ultra-pure water for using ultra-pure water, 0.2M successively, cross Q-Sepharose fast flow
Anion-exchange column is washed；
Loading：The sodium hyaluronate raw material 80mg of the biology extraction from cockscomb is dissolved in 100mL water and obtains sample solution, by sample
Product solution is with the flow velocity loading of 10.0mL/min to ratio of height to diameter for 18:1 Q-Sepharose fast flow anion-exchange columns
In, loading volume is 0.3 times of column volume；
(2) gradient elution：Gradient elution, flow velocity 10.0mL/ are carried out with the NaCl aqueous solutions of 0.4M, 0.6M and 1.0M
Min, the column volume that elution volume is 13 times collect the eluent of 0.4M NaCl aqueous solutions, for the solution containing sodium hyaluronate；
The eluent for collecting 1.0M NaCl aqueous solutions realizes separation for the solution containing heterozygosis chondroitin sulfate.
Embodiment 4
A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate, steps are as follows：
(1) it activates：The NaCl aqueous solutions and ultra-pure water for using ultra-pure water, 0.2M successively, cross Q-Sepharose fast flow
Anion-exchange column is washed；
Loading：The sodium hyaluronate raw material 40mg of the biology extraction from cockscomb is dissolved in 100mL water and obtains sample solution, by sample
Product solution is with the flow velocity loading of 7.5mL/min to ratio of height to diameter for 10:1 Q-Sepharose fast flow anion-exchange columns
In, loading volume is 0.15 times of column volume；
(2) gradient elution：Gradient elution, flow velocity 7.5mL/ are carried out with the NaCl aqueous solutions of 0.4M, 0.6M and 1.0M
Min, the column volume that elution volume is 12 times collect the eluent of 0.4M NaCl aqueous solutions, for the solution containing sodium hyaluronate；
The eluent of 0.6M NaCl aqueous solutions is collected, return to step (1) is detached again；Collect the elution of 1.0M NaCl aqueous solutions
Liquid realizes separation for the solution containing heterozygosis chondroitin sulfate.
(3) solution containing sodium hyaluronate obtained by step (2) is purified, specially：It was carried out after activated carbon adsorption
Filter, alcohol precipitation is dry, obtains sodium hyaluronate；Solution of the gained containing heterozygosis chondroitin sulfate is purified, specially：
After the solution concentrated by rotary evaporation containing heterozygosis chondroitin sulfate, dialysed 3 days with the molecular cut off MWCO bag filters for being 3500, so
After be freeze-dried, you can obtain impurity sample, obtain chondroitin sulfate.
Embodiment 5
A kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate, steps are as follows：
(1) loading：The sodium hyaluronate raw material 60mg of the biology extraction from cockscomb is dissolved in 100mL water and obtains sample solution,
By sample solution with the flow velocity loading of 8.5mL/min to ratio of height to diameter for 13:1 Q-Sepharose fast flow anion are handed over
It changes in column, the column volume that loading volume is 0.25 times；
(2) gradient elution：Gradient elution, flow velocity 8.5mL/ are carried out with the NaCl aqueous solutions of 0M, 0.4M, 0.6M and 1.0M
Min, the column volume that elution volume is 10 times collect the eluent of 0.6M NaCl aqueous solutions, and return to step (1) is divided again
From；The eluent for collecting 0.4M NaCl aqueous solutions, for the solution containing sodium hyaluronate；Collect the elution of 1.0M NaCl aqueous solutions
Liquid realizes separation for the solution containing heterozygosis chondroitin sulfate.
Comparative example 1
With differing only in for embodiment 1：Q-Sepharose fast flow anion-exchange columns are replaced with into DEAE the moon
Ion exchange column.
Comparative example 2
With differing only in for embodiment 1：Q-Sepharose fast flow anion-exchange columns are replaced with into dowex
1 × 2 strong alkalinity anion exchange column.
Comparative example 3
With differing only in for embodiment 1：Step (2) carries out gradient with the NaCl aqueous solutions of 0M, 0.4M, 0.8M and 1.0M
Elution.
Comparative example 4
With differing only in for embodiment 1：Step (2) carries out gradient with the NaCl aqueous solutions of 0M, 0.4M, 0.5M and 1.0M
Elution.
Embodiment 6
With differing only in for embodiment 1：A concentration of 0.2g/L of sample solution.
Embodiment 7
With differing only in for embodiment 1：A concentration of 2.0g/L of sample solution.
Embodiment 8
With differing only in for embodiment 1：Elution flow rate is 3.0mL/min.
Embodiment 9
With differing only in for embodiment 1：Elution flow rate is 13.0mL/min.
Each embodiment is arranged with the purity of the separating obtained sodium hyaluronate of comparative example and chondroitin sulfate in table 1.
Table 1
As shown in table 1, comparative examples 5 and comparative example 1~2 are it is found that Q-Sepharose fast used in the present invention
Other ion exchange columns, the purity of the sodium hyaluronate of gained significantly improve flow anion-exchange columns compared to the prior art,
And obtained heterozygosis chondroitin sulfate purity has high added value in 99.5wt% or more.And other ion exchange columns can only
Sodium hyaluronate is carried out a degree of purification, but the high efficiente callback of heterozygosis chondroitin sulfate is obtained almost without effect
The heterozygosis chondroitin sulfate amount arrived is few, and purity is very low, cannot directly apply.
Comparative examples 5 are with comparative example 3~4 it is found that the present invention passes through Q-Sepharose fast flow anion exchanges
Column need with suitable eluent gradient cooperate, the present invention successively use 0.4M, 0.6M and 1.0M NaCl aqueous solutions into
Row gradient elution, compared to the setting of other gradients, separative efficiency higher, products obtained therefrom purity has marked improvement.
5 embodiment 6~9 of comparative examples is it is found that the present invention passes through Q-Sepharose fast flow anion-exchange columns
It needs under the premise of cooperating with suitable eluent gradient, by the way that sample concentration, elution flow rate is arranged, is assisted mutually between parameter
Together, the purity of gained sodium hyaluronate and chondroitin sulfate is further increased.
Applicant states that the present invention illustrates detailed process equipment and the technological process of the present invention by above-described embodiment,
But the invention is not limited in above-mentioned detailed process equipment and technological processes, that is, it is above-mentioned detailed not mean that the present invention has to rely on
Process equipment and technological process could be implemented.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention,
The addition of equivalence replacement and auxiliary element to each raw material of product of the present invention, the selection etc. of concrete mode all fall within the present invention's
Within protection domain and the open scope.
Claims (10)
1. a kind of method of separation of glasses acid sodium and heterozygosis chondroitin sulfate, which is characterized in that include the following steps：
(1) loading：By sodium hyaluronate dissolution of raw material in water, sample solution is obtained；By sample solution loading to Q-Sepharose
In fast flow anion-exchange columns；
(2) gradient elution：Gradient elution is carried out with the NaCl aqueous solutions of 0.4M, 0.6M and 1.0M successively, collects 0.4M NaCl water
The eluent of solution, for the solution containing sodium hyaluronate；The eluent for collecting 1.0M NaCl aqueous solutions, to contain heterozygosis sulfuric acid
The solution of chondroitin.
2. the method for separation of glasses acid sodium and heterozygosis chondroitin sulfate as described in claim 1, which is characterized in that step (1)
The sodium hyaluronate raw material includes the sodium hyaluronate raw material of biological extraction method production；
Preferably, in step (1) described sample solution sodium hyaluronate raw material a concentration of 0.3~0.8g/L, preferably 0.4~
0.6g/L。
3. the method for separation of glasses acid sodium and heterozygosis chondroitin sulfate as claimed in claim 1 or 2, which is characterized in that step
(1) ratio of height to diameter of the Q-Sepharose fast flow anion-exchange columns is (10~20):1, preferably (10~15):1.
4. such as the method for claims 1 to 3 any one of them separation of glasses acid sodium and heterozygosis chondroitin sulfate, feature exists
Flow velocity when, step (1) described loading is 5.0~10.0mL/min, preferably 7.5~8.5mL/min；
Preferably, the column volume that loading volume is 0.1~0.3 times in the step (1).
5. such as the method for Claims 1 to 4 any one of them separation of glasses acid sodium and heterozygosis chondroitin sulfate, feature exists
In the step (1) further includes before：Activate Q-Sepharose fast flow anion-exchange columns；
Preferably, the activation specifically includes：Ultra-pure water, NaCl aqueous solutions and ultra-pure water, the excessively described Q-Sepharose are used successively
Fast flow anion-exchange columns are washed；
Preferably, a concentration of 0.2~0.6M of the NaCl aqueous solutions when activation.
6. such as the method for Claims 1 to 5 any one of them separation of glasses acid sodium and heterozygosis chondroitin sulfate, feature exists
In the column volume that the step (2) elution volume is 10~13 times；
Preferably, flow velocity when step (2) described gradient elution is 5.0~10.0mL/min, preferably 7.5~8.5mL/min.
7. such as the method for claim 1~6 any one of them separation of glasses acid sodium and heterozygosis chondroitin sulfate, feature exists
In carrying out gradient elution with the NaCl aqueous solutions of 0M, 0.4M, 0.6M and 1.0M successively in the step (2).
8. such as the method for claim 1~7 any one of them separation of glasses acid sodium and heterozygosis chondroitin sulfate, feature exists
In step (2) further includes：The eluent of 0.6M NaCl aqueous solutions is collected, return to step (1) is detached again.
9. such as the method for claim 1~8 any one of them separation of glasses acid sodium and heterozygosis chondroitin sulfate, feature exists
In the method further includes step (3)：Solution containing sodium hyaluronate obtained by step (2) is purified, Hyaluronic Acid is obtained
Sodium；
And/or purify the solution containing heterozygosis chondroitin sulfate obtained by step (2), obtain chondroitin sulfate.
10. such as the method for claim 1~9 any one of them separation of glasses acid sodium and heterozygosis chondroitin sulfate, feature exists
In including the following steps：
(1) it activates：The NaCl aqueous solutions and ultra-pure water of ultra-pure water, 0.2~0.6M, the excessively described Q-Sepharose fast are used successively
Flow anion-exchange columns are washed；
Loading：By the sample solution of a concentration of 0.3~0.8g/L with the flow velocity loading of 5.0~10.0mL/min to Q-
In Sepharose fast flow anion-exchange columns, loading volume is 0.1~0.3 times of column volume；
(2) gradient elution：Gradient elution is carried out with the NaCl aqueous solutions of 0M, 0.4M, 0.6M and 1.0M, flow velocity is 5.0~
10.0mL/min, the column volume that elution volume is 10~13 times collect the eluent of 0.4M NaCl aqueous solutions, to contain glass
The solution of sour sodium；The eluent for collecting 1.0M NaCl aqueous solutions, for the solution containing heterozygosis chondroitin sulfate；Collect 0.6M
The eluent of NaCl aqueous solutions, return to step (1) are detached again.
(3) it purifies：Solution containing sodium hyaluronate obtained by step (2) is purified, sodium hyaluronate is obtained；And/or by step
(2) solution of the gained containing heterozygosis chondroitin sulfate is purified, and obtains chondroitin sulfate.
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| CN109613132A (en) * | 2018-12-06 | 2019-04-12 | 上海景峰制药有限公司 | A kind of detection method of heterozygosis chondroitin sulfate |
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