CN109613157A - A kind of raw material heparin sodium for producing Enoxaparin Sodium and using - Google Patents
A kind of raw material heparin sodium for producing Enoxaparin Sodium and using Download PDFInfo
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- CN109613157A CN109613157A CN201811500028.6A CN201811500028A CN109613157A CN 109613157 A CN109613157 A CN 109613157A CN 201811500028 A CN201811500028 A CN 201811500028A CN 109613157 A CN109613157 A CN 109613157A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The present invention relates to heparin sodium technical field, in particular a kind of raw material heparin sodium for producing Enoxaparin Sodium and using prepares the heparin sodium raw materials of Enoxaparin Sodium using correlation coefficient process screening, the specific steps are that: heparin sodium enzymatic hydrolysis；Separation analysis；As a result it calculates.The present invention prepares the heparin sodium raw materials of Enoxaparin Sodium using correlation coefficient process screening, and correlation table and related figure can reflect correlation and its related direction between two variables.Related coefficient is the statistical indicator to reflect correlativity level of intimate between variable.Relationship more accurate quick between heparin sodium and Enoxaparin Sodium preparation is measured by related coefficient, the present invention using statistical correlation coefficient process is screened the disaccharides that can not only calculate known structure and including tetrose can also include the polysaccharide of unknown structure after degradation simultaneously, it is more comprehensively reliable, as a result the influence of testing staff and environment is not will receive, more scientific, accuracy rate is higher.
Description
Technical field
The present invention relates to heparin sodium technical field, specially a kind of raw material heparin sodium for producing Enoxaparin Sodium and using.
Background technique
Heparin sodium is mucopolysaccharide sulfuric acid ester anticoagulant, and heparin is that most effective in the world and quantity is maximum at present
Anticoagulation medicine is mainly used in cardiovascular and cerebrovascular disease and hemodialysis, wherein it is unique in hemodialysis
Effective specific medicament.Clinical application and studies have shown that heparin also have other a variety of biologies living in addition to blood coagulation resisting function
Property and clinical application, including effect for reducing blood fat, it is anti-in film smooth muscle cell (SMC) hyperplasia, promote the effects of fibrinolysis.
In addition, Enoxaparin Sodium is the drug for the major class antithrombotic being further processed by heparin bulk pharmaceutical chemicals as raw material, have more
For extensive clinical medicine use, become the treatment diseases such as Acute Venous thrombus and acute coronary artery syndrome (angina pectoris, myocardial infarction)
The choice drug of disease, and safety is higher, it is less side effects.
The naturally occurring mast cell of heparin sodium is mainly extracted from pig intestinal mucosa.Manufacturing enterprise is firstly the need of small from live pig
Heparin crude product is extracted and be made in intestinal mucosa, because containing foreign protein in heparin crude product, is not directly applicable clinical treatment, it need to be into
Heparin bulk pharmaceutical chemicals are processed into onestep extraction purifying.The heparin sodium crude extracted by chitterlings.
When extracting crude heparin sodium by pig intestinal mucosa, due to extracting method, each enterprise is multifarious, has enzymatic isolation method, has salt
Solution etc. is very big so as to cause crude heparin sodium quality difference；When purifying refined heparin sodium by crude heparin sodium, also have
Many methods, such as the sour alkali tune filtration method of tune, ion-exchange, salt solution, oxidizing process, method of purification and technological parameter
Difference has highly important influence to the quality of refined heparin sodium.And the quality of refined heparin sodium decides and uses its system
Make the quality of low molecular weight heparin Enoxaparin Sodium, so how to choose the refined heparin sodium that suitable Enoxaparin Sodium produces and uses
It is to put the problem of in the industry cycle encountering, the refined heparin sodium of selected quality, the matter for the Enoxaparin Sodium for being not only fabricated to it
It measures superior, and the yield of product also available raising, conversely, then Enoxaparin Sodium can't meet the quality requirement, and receives
Rate can also reduce.This specification is intended to provide the detection method of a kind of pair of refined heparin sodium, and method, which can ensure, uses this technology
The refined heparin sodium of selection preferably produces good Enoxaparin Sodium.
Made in a kind of production method of Enoxaparin Sodium of application publication number CN104193849A for Enoxaparin Sodium
The selection of refined heparin sodium limits, but only from several disaccharides and the percentage composition of tetrose structure to raw material heparin sodium into
Row screening, since the sugared structure species that heparin sodium includes are more, it is known that the disaccharides and tetrose percentage composition of structure are in 0.1%-
70% differs, and individual two sugared contents are very low, and the error of liquid phase same sample continuous sample introduction can only also be controlled 1.0% or so, because
The result of the detection of this percentage composition, which will receive instrumental sensitivity and the error of operator, to be influenced, and reproducibility is poor to be easy to appear
Error, selection result inaccuracy influence the preparation of low molecular sodium heparin, result in waste of resources and the unstability of technique.Simultaneously should
The disaccharides and tetrose that invention only applies known structure are calculated, and heparin sodium can generate a large amount of unknown knot after degradation
The bglii fragment of structure is not taken into account, and as a result spreadability is narrow, can not comprehensively react heparin sodium sugar structure and Enoxaparin Sodium
Relationship between preparation.
Summary of the invention
The purpose of the present invention is to provide a kind of raw material heparin sodiums for producing Enoxaparin Sodium and using, to solve above-mentioned background
The problem of being proposed in technology.The raw material heparin sodium that the production Enoxaparin Sodium uses is fast, prompt and comprehensive raw material screening method, preferably
Ground solves the problems, such as the screening for the raw material heparin sodium that Enoxaparin Sodium uses.Overall scientific of the present invention can not only guarantee product
Safety, validity, and can be avoided the waste of heparin sodium raw materials, improve production efficiency.
To achieve the above object, the invention provides the following technical scheme:
A kind of raw material heparin sodium for producing Enoxaparin Sodium and using, prepares low molecular sodium heparin using correlation coefficient process screening
Heparin sodium raw materials, the specific steps are that:
1) determination of heparin sodium raw material standard data: Enoxaparin Sodium is made with several batch heparin sodium raw materials, chooses and closes
5~10 batch of heparin sodium raw materials used in the Enoxaparin Sodium of lattice determines heparin sodium raw materials mark by digesting, separating analysis
Quasi- data；
2) heparin sodium raw materials to be measured are taken, by digesting, separating analysis, record heparin sodium sample map to be measured；
3) heparin sodium sample map to be measured is compared with heparin sodium raw material standard map, is screened using correlation coefficient process
Prepare the heparin sodium raw materials of low molecular sodium heparin.
Further, in step 1):
A. heparin sodium digests:
Heparin sodium bulk pharmaceutical chemicals are configured to aqueous solution, a certain amount of sodium acetate calcium solution are added thereto, to it after mixing
The mixing heparin enzyme solutions containing Heparinase I, Heparinase I I and Heparinase I II are added, gently overturning mixes, in 25 DEG C of water-baths
Constant temperature 48 hours or more, the enzymolysis liquid of heparin sodium is obtained, then a certain amount of sodium borohydride solution is added into enzymolysis liquid, is placed extremely
It is 4 hours few, the heparin sodium enzymolysis liquid after being restored；
B. separation analysis:
It is detected in heparin sodium enzymatic hydrolysis solution injection HPLC after being restored described in step a, records chromatogram, according to
The disaccharides of heparin sodium and the retention time of tetrose determine the type of the disaccharides and tetrose in heparin sodium to be measured, and according to 5~10 batches
The calculated by peak area average peak area of each of secondary heparin sodium raw materials spectrogram sugar, determines normal data.
Further, in step 2):
A1. heparin sodium digests:
Heparin sodium bulk pharmaceutical chemicals to be measured are configured to aqueous solution, a certain amount of sodium acetate calcium solution are added thereto, after mixing
The mixing heparin enzyme solutions containing Heparinase I, Heparinase I I and Heparinase I II are added to it, gently overturning mixes, in 25 DEG C of water
Constant temperature 48 hours or more in bath, the enzymolysis liquid of heparin sodium is obtained, then a certain amount of sodium borohydride solution is added into enzymolysis liquid, put
It sets at least 4 hours, the heparin sodium enzymolysis liquid after being restored；
B1. separation analysis:
It is detected in heparin sodium enzymatic hydrolysis solution injection HPLC after being restored described in step a1, records chromatogram, root
According to the disaccharides of heparin sodium and the retention time of tetrose, the type of the disaccharides and tetrose in heparin sodium to be measured is determined.
Further, step a, in step a1:
A2. heparin sodium aqua to be measured is prepared: precision measures heparin sodium 20mg, is placed in centrifuge tube, and 1mL water is added, and is vortexed
Dissolution, is made the solution of 20mg/mL；
B2. sodium acetate calcium solution is prepared: bovine serum albumin(BSA) 10mg, calcium acetate 32mg are taken, water 60mL is added to dissolve, it is on the rocks
580 μ L of acetic acid adjusts pH value to 7.0 with 2mol/l sodium hydroxide solution, and full dose is transferred in 100mL measuring bottle, is diluted with water to
Scale mixes, and is with after 0.45 μm of membrane filtration；
C. Heparinase I, II and III Compound mixed solution: taking heparin enzyme I, II and III is each appropriate, uses the phosphorus of pH7.0 respectively
Sour potassium buffer dissolves and is made the solution in every 1mL containing 0.4IU, take 3 kinds of heparin enzyme solutions respectively, in 1: 1: 1 ratio etc.
Volume mixture, shake up to get；
D. sodium borohydride solution is prepared: taking sodium borohydride 12mg, 400 μ L of water is added to make to dissolve, be vortexed and mix to get use is faced
Brand-new；
E. heparin sodium enzyme digestion reaction: 20 μ L of solution made from a step is taken, 70 μ L of calcium acetate solution and heparin are separately added into
100 μ L of enzyme I, II and III mixed liquor, gently overturning mixes, and constant temperature 48 hours or more in 25 DEG C of water-baths, obtains heparin sodium enzymatic hydrolysis
Liquid；
F. enzymolysis liquid reduction reaction: taking each 60 μ L of enzymolysis liquid made from step e, be separately added into 10 μ L of sodium borohydride solution,
It mixes, places at least 4 hours, the heparin sodium enzymolysis liquid after being restored.
Further, kaliumphosphate buffer is added in step f, prepares are as follows: take potassium dihydrogen phosphate 68mg, bovine serum albumin
White 10mg adds water 30mL to dissolve, and adjusts pH value to 7.0 with potassium hydroxide test solution, full dose is transferred in 50mL measuring bottle, is diluted with water
To scale, shake up, with after 0.45 μm of membrane filtration to obtain the final product.
Further, step b, in step b1:
Testing conditions are as follows: chromatographic column is strong anion exchange resin, and 50 DEG C of column temperature, flow velocity is 0.8mL per minute；With height
Sodium chlorate sodium dihydrogen phosphate is that Mobile phase B carries out gradient elution, gradient such as following table；
Further, step b, in step b1:
A3. mobile phase is prepared: mobile phase A: precision weighs sodium dihydrogen phosphate 0.280g, adds water 950m to make to dissolve, uses phosphoric acid
PH value is adjusted to 3.0, be diluted with water to 1000mL to get；
Mobile phase B: precision weighs sodium perchlorate 140g, and mobile phase A 950mL is added to make to dissolve, extremely with phosphorus acid for adjusting pH value
3.0, with mobile phase A be diluted to 1000mL to get；
B3. chromatographiccondition: instrument is high performance liquid chromatograph, and chromatographic column is strong anion exchange resin, detector
For UV detector, 50 DEG C of column temperature, flow velocity is 0.8mL per minute；Detection wavelength is 234nm；10 μ L of sample volume, with mobile phase A
Gradient elution is carried out with Mobile phase B, gradient see the table below:
C1. separation parsing: injecting liquid chromatograph for the heparin sodium enzymolysis liquid after the reduction obtained in b3 respectively, records color
Spectrogram.
Further, in step 3):
According to liquid chromatogram obtained in step b, step b1, related coefficient is calculated by following formula, passes through phase relation
Number can filter out the heparin sodium raw materials that suitable Enoxaparin Sodium uses；
Further, in step 3):
Ai is the area value at i-th of peak in map to be measured, and bi is that (5-10 batch can be made normal data, that is, control map
The map of the standby heparin sodium raw materials of high quality Enoxaparin Sodium out) in i-th of peak centre plane product value,It is each in map to be measured
The average value of peak area,For the average value of peak area each in standard diagram, n is the number of chromatographic peak.
Further, in step 3): the related coefficient of sample to be tested is greater than 0.95.
Compared with prior art, the beneficial effects of the present invention are:
The present invention prepares the heparin sodium raw materials of low molecular sodium heparin using correlation coefficient process screening, and correlation table and related figure can
Reflect the correlation and its related direction between two variables.Related coefficient is that correlativity is close between variable to reflect
The statistical indicator of degree.Relationship more accurate quick between heparin sodium and Enoxaparin Sodium preparation is measured by related coefficient,
The statistical correlation coefficient process of present invention use simultaneously, which is screened, not only can calculate the disaccharides of known structure and tetrose may be used also
With will be more comprehensively reliable including the polysaccharide of unknown structure is included after degradation, testing staff and environment as a result not will receive
It influences, more scientific, accuracy rate is higher.
Detailed description of the invention
Fig. 1 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 1st crowd.
Fig. 2 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 2nd crowd.
Fig. 3 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 3rd crowd.
Fig. 4 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 4th crowd.
Fig. 5 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 5th crowd.
Fig. 6 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 6th crowd.
Fig. 7 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 7th crowd.
Fig. 8 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 8th crowd.
Fig. 9 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 9th crowd.
Figure 10 is liquid chromatogram after the heparin sodium sample enzymatic hydrolysis reduction to be measured of the present invention the 10th crowd.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment, the raw material heparin sodium that raw material heparin sodium, the nadroparin calcium used such as Dalteparin Sodium uses belong to protection of the present invention
Range.
Fig. 1-Figure 10 is please referred to, the present invention provides a kind of technical solution:
A kind of raw material heparin sodium for producing Enoxaparin Sodium and using, is achieved by the following measures:
The detection and calculating of polysaccharide structures
1) heparin sodium digests:
Heparin sodium bulk pharmaceutical chemicals are configured to aqueous solution, a certain amount of sodium acetate calcium solution (pH7.0) is added thereto, are mixed
The mixing heparin enzyme solutions containing Heparinase I, Heparinase I I and Heparinase I II are added in even backward its, and gently overturning mixes, 25
Constant temperature 48 hours or more in DEG C water-bath, the enzymolysis liquid of heparin sodium is obtained, then that a certain amount of sodium borohydride is added into enzymolysis liquid is molten
Liquid places at least 4 hours (solution is stablized in 48 hours at room temperature), the heparin sodium enzymolysis liquid after being restored.
2) separation analysis:
1) it is detected in the heparin sodium enzymatic hydrolysis solution injection HPLC after restoring described in, chromatogram is recorded, according to liver
The disaccharides of plain sodium and the retention time of tetrose determine the type of the disaccharides and tetrose in heparin sodium to be measured, and according in spectrogram
The related coefficient of the calculated by peak area similarity of each sugar；
Wherein testing conditions are as follows: chromatographic column is strong anion exchange resin, and 50 DEG C of column temperature, flow velocity is 0.8mL per minute；With
Sodium perchlorate sodium dihydrogen phosphate is that Mobile phase B carries out gradient elution, gradient such as the following table 1.
1 gradient elution table of table
3) result calculates:
The liquid chromatogram according to obtained in 2) calculates related coefficient by following formula, can be sieved by related coefficient
Select the heparin sodium raw materials that suitable low molecular weight heparin uses.
Wherein ai is the area value at i-th of peak in map to be measured, and bi is normal data, that is, control map (5-10 batch
The map of the heparin sodium raw materials of high quality Enoxaparin Sodium can be prepared) in i-th of peak centre plane product value,For to mapping
The average value of each peak area in spectrum,For the average value of peak area each in standard diagram, n is the number of chromatographic peak.
When the related coefficient of sample to be tested is greater than 0.95, illustrate the sugared structure composition and heparin sodium mark of heparin sodium raw materials to be measured
The sugared structure composition of quasi- sample largely approaches, and can be used as the reserve materials for preparing low molecular sodium heparin.
Embodiment 1
1) heparin sodium aqua is prepared: precision measures sample to be tested heparin sodium and each 20mg of heparin sodium standard, is placed in centrifuge tube
It is interior, 1mL water is added, be vortexed dissolution, and the solution of 20mg/mL is made.
2) sodium acetate calcium solution is prepared: being taken bovine serum albumin(BSA) 10mg, calcium acetate 32mg, is added water 60mL to dissolve, vinegar on the rocks
580 μ L of acid adjust pH value to 7.0 with 2mol/l sodium hydroxide solution, and full dose is transferred in 100mL measuring bottle, is diluted with water to quarter
Degree mixes, and is with after 0.45 μm of membrane filtration.
3) kaliumphosphate buffer is prepared: taking potassium dihydrogen phosphate 68mg, bovine serum albumin(BSA) 10mg, water 30mL is added to dissolve, use
Potassium hydroxide test solution adjusts pH value to 7.0, and full dose is transferred in 50mL measuring bottle, is diluted with water to scale, is shaken up, and is filtered with 0.45 μm
After film filtering to obtain the final product.
4) Heparinase I, II and III Compound mixed solution: taking heparin enzyme I, II and III is each appropriate, is buffered respectively with potassium phosphate
Liquid (pH7.0) dissolves and is made the solution in every 1mL containing 0.4IU (20 DEG C can store 3 months), takes 3 kinds of heparinases molten respectively
Liquid is mixed in equal volume in 1: 1: 1 ratio, shake up to get.
5) sodium borohydride solution is prepared: taking sodium borohydride 12mg, 400 μ L of water is added to make to dissolve, be vortexed and mix to get use is faced
Brand-new.
6) heparin sodium enzyme digestion reaction: take 1) made from each 20 μ L of solution, be separately added into 70 μ L of calcium acetate solution and heparinase
I, 100 μ L of II and III mixed liquor, gently overturning mixes, and constant temperature 48 hours or more in 25 DEG C of water-baths, obtains heparin sodium enzymatic hydrolysis
Liquid.
7) enzymolysis liquid reduction reaction: take 6) made from each 60 μ L of enzymolysis liquid, be separately added into 10 μ L of sodium borohydride solution, mix
It is even, at least 4 hours (solution is stablized in 48 hours at room temperature) are placed, the heparin sodium enzymolysis liquid after being restored.
8) mobile phase is prepared: mobile phase A-precision weighs sodium dihydrogen phosphate 0.280g, adds water 950mL to make to dissolve, uses phosphoric acid
PH value is adjusted to 3.0, be diluted with water to 1000mL to get.Mobile phase B-precision weighs sodium perchlorate 140g, adds mobile phase A
950mL makes to dissolve, with phosphorus acid for adjusting pH value to 3.0, with mobile phase A be diluted to 1000mL to get.
9) chromatographiccondition: instrument is high performance liquid chromatograph, and chromatographic column is strong anion exchange resin, and detector is
UV detector, 50 DEG C of column temperature, flow velocity is 0.8mL per minute；Detection wavelength is 234nm；10 μ L of sample volume, with mobile phase A and
Mobile phase B carries out gradient elution, and gradient is shown in Table 2.
2 gradient elution table of table
10) separation parsing: the heparin sodium enzymolysis liquid after the reduction 9) obtained is injected into liquid chromatograph respectively, records chromatography
Figure.
11) result calculates: according to the relative retention time of disaccharides and tetrose such as table 3, determining disaccharides and tetrose in chromatogram
Position, count spectrogram in each peak peak area, the related coefficient of sample to be tested and standard items is calculated according to the following equation.Meter
It is as follows to calculate formula
Wherein ai is the area value at i-th of peak in map to be measured, and bi is that (5-10 batch can for normal data, that is, control map
To prepare the map of the heparin sodium raw materials of high quality Enoxaparin Sodium) in i-th of peak centre plane product value,For map to be measured
In each peak area average value,For the average value of peak area each in standard diagram, n is the number of chromatographic peak.It is related in spectrogram
The appearance time at peak such as following table 4.
Polysaccharide structures appearance time after table 4 restores
Statistics heparin sodium normal data (determines) 5 batch numbers according to the heparin sodium raw materials for preparing qualified Enoxaparin Sodium
According to after being averaged as calculate in standard sample spectrogram peak area, have chosen the heparin sodium raw materials of 10 batches of different batches respectively
Carry out experiment and will be according to step 1) -11) carry out enzymolysis processing and liquid-phase chromatographic analysis, calculate and the phase of heparin sodium normal data
Relationship number.And 10 batch heparin sodium raw materials of experiment are carried out to the preparation of Enoxaparin Sodium according to specific preparation process, to according to promise
Heparin sodium finished product carries out quality inspection, verifies the related coefficient of different heparin sodium raw materials and the relationship of Enoxaparin Sodium quality, from
And heparin sodium raw materials are screened.
As a result due to the big calculating for influencing related coefficient of the peak area of wherein Δ I S in calculating, and the heparin of each batch
The difference of its content is little in sodium, therefore removes it during calculating related coefficient, and content is lower than during the experiment
0.1% each peak is not counted in data statistics.The following table 5 is the correlation calculations knot after the heparin sodium of 10 batches digests with standard items
Fruit, table 6 are the relationship of related coefficient and finished product Enoxaparin Sodium quality.
5 10 batches of heparin sodium raw materials relevance detection results of table
6 10 batches of heparin sodium raw materials of table and Enoxaparin Sodium relationship between quality
The sugared structure composition of heparin sodium bulk pharmaceutical chemicals will have a direct impact on the molecular weight of finished product Enoxaparin Sodium, potency and 1,6-
The content of anhydro derivatives structure.Work as the phase of heparin sodium to be measured and heparin sodium raw material standard data it can be seen from experimental result
When relationship number reaches 0.95 (sample 1-7) or more, Enoxaparin Sodium prepared therefrom is through detection molecules amount, potency and 1,6- dehydration
The content of derivative is met the requirements of the standard, and related coefficient is closer to 1, by sample to be tested preparation Enoxaparin Sodium it is each
Quality index and Enoxaparin Sodium standard items are closer, and the Enoxaparin Sodium quality of preparation is better；On the contrary, working as the phase of sample to be tested
When relationship number is less than 0.95 (sample 8-10), Enoxaparin Sodium prepared therefrom does not meet quality criteria requirements, and related coefficient is got over
Small, each quality index of finished product Enoxaparin Sodium and the gap of Enoxaparin Sodium standard items are bigger, the Enoxaparin Sodium matter of preparation
It is poorer to measure.
Therefore, can be used for preparing when the related coefficient of heparin sodium raw materials to be measured and heparin sodium standard items reaches 0.95 or more
Enoxaparin Sodium, as far as possible selection related coefficient and 1 close heparin sodium raw materials, the quality of the Enoxaparin Sodium of preparation and according to promise liver
Plain sodium standard items are closer.Similarly, this related coefficient raw material screening method can be applied to other low molecular sodium heparin (dalteparinSodium
Sodium, nadroparin calcium etc.) it prepares in the application and preparations of polysaccharide structures such as series and hyaluronic acid.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (10)
1. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using, which is characterized in that using correlation coefficient process screening preparation according to
The heparin sodium raw materials of promise heparin sodium；
1) determination of heparin sodium raw material standard data: being made Enoxaparin Sodium with several batch heparin sodium raw materials, chooses qualification
5~10 batch of heparin sodium raw materials used in Enoxaparin Sodium determines heparin sodium raw material standard number by digesting, separating analysis
According to；
2) heparin sodium raw materials to be measured are taken, by digesting, separating analysis, record heparin sodium sample map to be measured；
3) heparin sodium sample map to be measured is compared with heparin sodium raw material standard map, is screened and is prepared using correlation coefficient process
The heparin sodium raw materials of low molecular sodium heparin.
2. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 1, which is characterized in that step 1)
In:
A. heparin sodium digests:
Heparin sodium raw materials are configured to aqueous solution, a certain amount of sodium acetate calcium solution is added thereto, are contained after mixing to its addition
There are the mixing heparin enzyme solutions of Heparinase I, Heparinase I I and Heparinase I II, gently overturning mixes, the constant temperature 48 in 25 DEG C of water-baths
Hour or more, the enzymolysis liquid of heparin sodium is obtained, then a certain amount of sodium borohydride solution is added into enzymolysis liquid, it is small to place at least 4
When, the heparin sodium enzymolysis liquid after being restored；
B. separation analysis:
It is detected in heparin sodium enzymatic hydrolysis solution injection HPLC after being restored described in step a, chromatogram is recorded, according to heparin
The disaccharides of sodium and the retention time of tetrose determine the type of the disaccharides and tetrose in heparin sodium to be measured, and according to 5~10 batch livers
The calculated by peak area average peak area of each of plain sodium raw materials spectrogram sugar, determines normal data.
3. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 2, which is characterized in that step 2)
In:
A1. heparin sodium digests:
Heparin sodium raw materials to be measured are configured to aqueous solution, a certain amount of sodium acetate calcium solution, Xiang Qijia after mixing are added thereto
Enter the mixing heparin enzyme solutions containing Heparinase I, Heparinase I I and Heparinase I II, gently overturning mixes, permanent in 25 DEG C of water-baths
Temperature 48 hours or more, the enzymolysis liquid of heparin sodium is obtained, then a certain amount of sodium borohydride solution is added into enzymolysis liquid, placed at least
4 hours, the heparin sodium enzymolysis liquid after being restored；
B1. separation analysis:
It is detected in heparin sodium enzymatic hydrolysis solution injection HPLC after being restored described in step a1, chromatogram is recorded, according to liver
The disaccharides of plain sodium and the retention time of tetrose determine the type of the disaccharides and tetrose in heparin sodium to be measured.
4. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 3, which is characterized in that step 3)
In:
According to liquid chromatogram obtained in step b, step b1, related coefficient is calculated by following formula, is by related coefficient
The heparin sodium raw materials that suitable Enoxaparin Sodium uses can be filtered out；
5. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 3, which is characterized in that step a,
In step a1:
A2. heparin sodium aqua is prepared: precision measures heparin sodium 20mg, is placed in centrifuge tube, and 1mL water is added, and be vortexed dissolution, is made
The solution of 20mg/mL；
B2. sodium acetate calcium solution is prepared: being taken bovine serum albumin(BSA) 10mg, calcium acetate 32mg, is added water 60mL to dissolve, acetic acid on the rocks
580 μ L, with 2mol/l sodium hydroxide solution adjusting pH value to 7.0, full dose is transferred in 100mL measuring bottle, is diluted with water to scale,
It mixes, is with after 0.45 μm of membrane filtration；
C. Heparinase I, II and III Compound mixed solution: taking heparin enzyme I, II and III is each appropriate, slow with the potassium phosphate of pH7.0 respectively
Fliud flushing dissolves and is made the solution in every 1mL containing 0.4IU, take 3 kinds of heparin enzyme solutions respectively, mixes in equal volume in 1: 1: 1 ratio
Close, shake up to get；
D. sodium borohydride solution is prepared: taking sodium borohydride 12mg, 400 μ L of water is added to make to dissolve, be vortexed and mix to get facing and using brand-new；
E. heparin sodium enzyme digestion reaction: 20 μ L of solution made from a step is taken, 70 μ L of calcium acetate solution and Heparinase I, II are separately added into
With 100 μ L of III mixed liquor, gently overturning is mixed, and constant temperature 48 hours or more in 25 DEG C of water-baths, obtains heparin sodium enzymolysis liquid；
F. enzymolysis liquid reduction reaction: taking each 60 μ L of enzymolysis liquid made from step e, be separately added into 10 μ L of sodium borohydride solution, mixes,
It places at least 4 hours, the heparin sodium enzymolysis liquid after being restored.
6. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 5, which is characterized in that step f
Middle addition kaliumphosphate buffer is prepared are as follows: takes potassium dihydrogen phosphate 68mg, bovine serum albumin(BSA) 10mg, water 30mL is added to dissolve, use
Potassium hydroxide test solution adjusts pH value to 7.0, and full dose is transferred in 50mL measuring bottle, is diluted with water to scale, is shaken up, and is filtered with 0.45 μm
After film filtering to obtain the final product.
7. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 3 or 4, which is characterized in that step
In rapid b, step b1:
Testing conditions are as follows: chromatographic column is strong anion exchange resin, and 50 DEG C of column temperature, flow velocity is 0.8mL per minute；With sodium perchlorate
Sodium dihydrogen phosphate is that Mobile phase B carries out gradient elution, gradient such as following table；
8. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 7, which is characterized in that step b,
In step b1:
A3. mobile phase is prepared: mobile phase A: precision weighs sodium dihydrogen phosphate 0.280g, adds water 950mL to make to dissolve, is adjusted with phosphoric acid
PH value to 3.0, be diluted with water to 1000mL to get；
Mobile phase B: precision weighs sodium perchlorate 140g, and mobile phase A 950mL is added to make to dissolve, and with phosphorus acid for adjusting pH value to 3.0, uses
Mobile phase A be diluted to 1000mL to get；
B3. chromatographiccondition: instrument is high performance liquid chromatograph, and chromatographic column is strong anion exchange resin, and detector is purple
External detector, 50 DEG C of column temperature, flow velocity is 0.8mL per minute；Detection wavelength is 234nm；10 μ L of sample volume, with mobile phase A and stream
Dynamic phase B carries out gradient elution, and gradient see the table below:
C1. the heparin sodium enzymolysis liquid after the reduction obtained in b, step b1 separation parsing: is injected into liquid chromatograph, record respectively
Chromatogram.
9. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 4, which is characterized in that step 3)
In:
Ai is the area value at i-th of peak in map to be measured, and bi is the average area at i-th of peak in normal data, that is, control data
Value,For the average value of each peak area in map to be measured,For the average value of peak area each in normal data, n is the number of chromatographic peak
Mesh.
10. a kind of raw material heparin sodium for producing Enoxaparin Sodium and using according to claim 8, which is characterized in that step
3) in: the related coefficient of sample to be tested is greater than 0.95.
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