CN104877024A - Crude drug bivalirudin purification process - Google Patents
Crude drug bivalirudin purification process Download PDFInfo
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- CN104877024A CN104877024A CN201510368448.3A CN201510368448A CN104877024A CN 104877024 A CN104877024 A CN 104877024A CN 201510368448 A CN201510368448 A CN 201510368448A CN 104877024 A CN104877024 A CN 104877024A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
Abstract
The invention belongs to the field of pharmaceutical synthesis and particularly relates to a crude drug bivalirudin purification process. According to the bivalirudin purification and drying process, two purification systems are combined to complete bivalirudin purification, a chromatographic system for first-time purification (coarse purification) consists of a mobile phase A, namely 0.025%-0.2% of heptafluorobutyric acid solution, and a mobile phase B, a chromatographic column filler is an octadecyl silane bonded silica gel filler with the particle diameter ranging from 45 microns to 75 microns, the purity can be up to 99.8% after gradient elution, a chromatographic system for second-time purification (refine purification) consists of a mobile phase A, namely 0.01%-0.05% of trifluoroacetic acid solution, and a mobile phase B, a chromatographic column filler is an octadecyl silane bonded silica gel filler with 10-microns particle diameter, and the purity of a concentrated solution can be up to 99.8% after gradient elution and twice purification.
Description
Technical field
The invention belongs to pharmaceutical synthesis field, be specifically related to a kind of purifying process of Angiomax bulk drug.
Background technology
Angiomax (Bivalirudin) is a kind of direct thrombin inhibitor (DTI), comes from hirudin derivative, is a synthesis small peptide be made up of 20 amino acid.Angiomax is a kind of reversible inhibitor of effective, highly selective, can not only suppress the zymoplasm of sequestered and mating type, can also suppress by the platelet activation of thrombin-mediated and gathering.Angiomax drug effect is fast, the transformation period is short, in body not with plasma proteins and erythrocyte binding, to heparin-induced thrombocytopenia to Cheng with heparin-induced Xue bolt Xing ?thrombopenia syndromes (HIT/HITTS) there is not risk, in addition, in treatment, do not need in conjunction with cofactors such as antithrombins, also need not activate thrombocyte, these features make Angiomax become a desirable heparin substitute.
Angiomax (being also called Bivalirudin) is a kind of polypeptide by 28 seed amino acid chemosynthesis.Its structural formula is as follows:
D-Phe-L-Pro-L-Arg-L-Pro-Gly-Gly-Gly-Gly-L-Asn-Gly-L-Asp-L-Phe-L-Glu-L-Glu-L-Ile-L-Pro-L-Glu-L-Glu-L-Tyr-L-Leu
In December, 2000, the injectable dosage forms of this medicine of Medicines company of U.S. exploitation is gone on the market in the U.S., and commodity are called Angiomax, general Bivalirudin by name, and Chinese Angiomax by name, listing formulation is lyophilized injectable powder, and specification is that 250mg/ props up.
1 ~ February in 2005, Medicines company the Subsidiary Company Nycomed in Europe Angiox (Bivalirudin is in the trade(brand)name in Europe) Austria, Denmark.Finland, Germany, Switzerland and Britain's listing, intervene patient for percutaneous coronary artery.The specification of its listing is the aseptic freeze-dried powder of 250mg/ bottle.Angiomax is used for intravenous injection and instillation.
The method preparing Angiomax has bibliographical information, Solid-phase synthesis peptides and liquid phase synthesizing method can be adopted to prepare the thick peptide of Angiomax (according to related substance detection method in national drug standards YBH02852011, foreign matter content is above standard the Angiomax crude product of middle regulation, the thick peptide of Angiomax can be defined as), but for the purifying of the thick peptide of Angiomax, only there is bibliographical information few in number.
Chinese patent CN200910028793.7 only describes and adopts just peptide and the lyophilize of HPLC method purifying Angiomax, have no detailed purifying process and the description of freeze-dry process, and product purity only can reach 98.5%, must not regulation more than 1.5% according to related substance in national drug standards YBH02852011, the Angiomax bulk drug of preparation is close to limit of impurities, in validity period 2 years, standing storage is easy to cause Related substances separation item defective.
The peptide that the method that Chinese patent CN200980152943.3 discloses the first peptide of a kind of purifying Angiomax adopts preparation scale HPLC rough described in purifying on the anti-phase stationary phase of C18.The first step, adopts peptide rough described in ammonium acetate/water/acetonihile gradient elution purifying, then at second step, adopts trifluoroacetic acid/water/acetonihile gradient elution.But the condition of undeclared concrete purifying, cannot reappear its experimental result in literary composition.
The purification process of Angiomax crude product is example 8 describes: take Angiomax crude powder in Chinese patent application CN201110170669.1 specification sheets, crude product is added to (about 20ml water/g crude product) in purified water, stir lower dropping weak ammonia and adjust pH, pH is controlled ~ 5.0, solution 0.45 μm of mixing filtering with microporous membrane, standby purifying is used.High performance liquid chromatography is adopted to carry out purifying, purifying chromatograph packing material is the anti-phase C18 of 10 μm, flow phase system be 0.1%TFA/ Shui Rong Ye ?0.1%TFA/ acetonitrile solution, the column flow rate of 77mm*250mm is 90ml/min, adopts gradient system wash-out, circulation sample introduction purifying, getting crude product solution is splined in chromatographic column, start moving phase wash-out, after collection main peak boils off acetonitrile, obtain Angiomax purify intermediates concentrated solution.Get Angiomax purify intermediates concentrated solution, filter for subsequent use with 0.45 μm of filter membrane.High performance liquid chromatography is adopted to carry out changing salt, flow phase system be 0.1%TFA/ Shui Rong Ye ?acetonitrile, purifying chromatograph packing material is the column flow rate of anti-phase C18, the 77mm*250mm of 10 μm is 90ml/min (according to the chromatographic column of different size, can adjust corresponding flow velocity).Adopt gradient elution, quadrat method in circulation, is splined in chromatographic column, start moving phase wash-out, gather collection of illustrative plates, the change of observation optical density, collection is changed salt main peak and is used and analyzes Liquid Detection purity, merging and change salt main peak solution, being less than concentrating under reduced pressure under 40 DEG C of water bath condition, boiling off most of acetonitrile with Rotary Evaporators, obtain Angiomax trifluoroacetic acid aqueous solution, lyophilize, obtains product 608g, and total recovery is 55.8%.Only adopting a kind of chromatogram purification system in this patent, and do not announce the embodiment of gradient elution, needing by repeatedly collecting the Angiomax solution meeting related substance in national drug standards YBH02852011, concentrated after merging, freeze-drying.The method can cause Angiomax bulk drug in Chunhua process, because only have a kind of moving phase to form carry out wash-out, the resolution of impurity and Angiomax cannot be guaranteed, need repeatedly repeatedly to collect the higher product solution of purity, loss greatly, is unfavorable for the bulk drug of preparation of industrialization feather weight.
Chinese patent application CN201310045298.3 discloses the purification process of Angiomax: gained crude product is adopted RPLC gradient elution purifies and separates, take octadecylsilane chemically bonded silica as stationary phase, eluent w (TFA): V (acetonitrile): V (water)=1:250:750 ~ 1:400:600, flow velocity 500ml/min, determined wavelength 214nm.Revolve the elutriant steaming and collect, obtain Angiomax 0.54kg after lyophilize, purity 99.8%, overall yield 37%.The method equally only adopts a kind of chromatogram purification system, and gradient has no detailed description, cannot reappear product preparation process.
The purification process of Chinese patent application CN201310188441.4 embodiment 10, Angiomax crude product: take Angiomax crude powder 8.0g prepared by embodiment 9, adopts acetonitrile solution to dissolve, solution 0.45 μm of filtering with microporous membrane, for subsequent use.High performance liquid chromatography carries out condition during purifying, chromatographic column: with the octadecylsilane chemically bonded silica of 10um for stationary phase, and pillar diameter and length are: 50mm × 250mm; Moving phase: 0.1%TFA/ Shui Rong Ye ?0.1%TFA/ acetonitrile solution; The flow velocity 60ml/min of wash-out; Adopt gradient elution, circulation input mode loading.The sample solution of above-mentioned process is splined in chromatographic column, starts moving phase wash-out, collect main peak and use and analyze Liquid Detection purity, merge main peak solution, being less than concentrating under reduced pressure under 40 DEG C of water bath condition, boiling off most of acetonitrile with Rotary Evaporators, obtaining Angiomax trifluoroacetic acid salts solution.Lyophilize obtains product 2.48g, total recovery 31%, and product purity is 99.6%, Angiomax [+Gly] impurity, Angiomax [?Gly] impurity is all less than 0.1%.Obtain Angiomax sterling.The method equally only adopts a kind of chromatogram purification system, and gradient has no detailed description, and preparative-scale is only 2.5g.Sample feasibility for production feather weight is on the knees of the gods.
Chinese patent application CN201310210329.6 embodiment 12 discloses the preparation method of Angiomax essence peptide trifluoroacetate: take 200g according to after the mixed solvent 5000mL dissolving of the thick peptide of Angiomax 25% acetonitrile+25%DMSO+50% water obtained described in embodiment nine, adopt Waters2545RP ?HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 × 250mm, column temperature is 50 DEG C, conventional 0.1%TFA/ acetonitrile mobile phase purifying, collect object peak cut, obtain purity and be greater than 98.5% smart peptide.Smart peptide solution is adopted Waters2545RP ?HPLC system, chromatographic column is the anti-phase C8 post of 50 × 250mm, 0.1% trifluoracetic acid/acetonitrile mobile phase turns salt, collect object peak cut, concentrated by rotary evaporation, freeze-drying obtains Angiomax trifluoroacetate essence peptide 79.8g, HPLC purity 99.53%, purification yield 81.4%, total recovery 35.9%.The method only adopts a kind of chromatogram purification system, gradient has no detailed description, in purge process, thick peptide dissolves DMSO (dimethyl sulfoxide (DMSO)) solution employing 25%, and for preparing the bulk drug of injection use, the control carrying out residual solvent for DMSO is needed in the quality control system of bulk drug, add cost and the cycle of detection, the HPLC purity 99.53% of product, always must not mix more than 1.5% according to related substance in national drug standards YBH02852011, known impurities ASP
9?Angiomax and D ?Phe
12?Angiomax must not regulation more than 0.5%, this Angiomax bulk drug prepared is close to 1/3 of total impurities limit, and in validity period 2 years, standing storage is easy to quality problems such as causing Related substances separation item defective.
The purifying process of the bulk drug of above-mentioned Angiomax, product yield, production capacity are all on the low side or purity is on the low side, and freeze-dry process cannot guarantee easily known impurities ASP during sample freeze-drying
9?Angiomax do not raise, be therefore unfavorable for suitability for industrialized production feather weight, the Angiomax bulk drug that Related substances separation item is qualified can be guaranteed before the deadline.
For the series of problems that existing Angiomax bulk drug purifying process exists, the invention provides a kind of purification process of Angiomax.
Summary of the invention
The object of the present invention is to provide a kind of purification process of Angiomax.The Angiomax prepared by the method has that yield is high, purity high.
The purifying and the freeze-dry process that the present invention is directed to Angiomax comprise the purifying having combined Angiomax with 2 kinds of purification system, the chromatographic of first time purifying (slightly pure) is by mobile phase A: 0.025 to 0.2% hyptafluorobutyric acid solution and Mobile phase B form, chromatographic column filler is the octadecylsilane chemically bonded silica filler of particle diameter 45 μm ~ 75 μm, after gradient elution, purity can reach 99.8%, the chromatographic of second time purifying (consummate) is by mobile phase A: 0.01 ~ 0.05% trifluoroacetic acid solution and Mobile phase B form, chromatographic column filler is the octadecylsilane chemically bonded silica filler of particle diameter 10 μm, gradient elution, solution after twice purifying concentrates, the purity of concentrated solution can reach 99.90%, according to the freeze-drying curve freeze-drying drafted after concentrated solution, to guarantee during freeze-drying known impurities ASP9 ?Angiomax do not change, the purity of bulk drug can be controlled in more than 99.9%.The industrial production scale that 10kg/ criticizes can be completed.
Concrete, purification process of the present invention, comprises the following steps:
(1) Angiomax crude product is got, add purified water dissolve and be mixed with crude product solution, subsequently crude product solution is injected high performance liquid chromatograph and carry out purifying, purification condition is: chromatographic is by mobile phase A: 0.025 to 0.2% hyptafluorobutyric acid solution and Mobile phase B form, chromatographic column filler is the octadecylsilane chemically bonded silica filler of particle diameter 45 μm ~ 75 μm, after gradient elution, purity can reach 99.8%
(2) the thick pure solution of the Angiomax upper step prepared, be injected into preparative high performance liquid chromatography instrument and carry out second time purifying, purification condition: chromatographic is by mobile phase A: 0.01 ~ 0.05% trifluoroacetic acid solution and Mobile phase B form, chromatographic column filler is the octadecylsilane chemically bonded silica filler of particle diameter 10 μm, gradient elution, solution after twice purifying concentrates, and the purity of concentrated solution can reach 99.90%
(3) concentrated solution freeze-drying, to obtain final product.
Wherein, high-efficient liquid phase chromatogram condition in step (1):
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: C
18, post footpath Φ 15cm,
Chromatographic column filler: 45 μm ~ 75 μm, 100A
Mobile phase A: 0.025 to 0.2% hyptafluorobutyric acid solution, Mobile phase B: acetonitrile,
Wavelength: 230nm,
Flow velocity: 400ml/ minute,
Gradient:
Sample size :≤50g/ pin
Circulation loading wash-out operates, and collects main peak solution,
And sample standard: main peak purity >=99.6%.
Get the solution of the Angiomax obtained through purifying in step (1), add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve steam to vacuum tightness Yue ? below 0.09Mpa, obtain the thick pure solution of Angiomax.
High-efficient liquid phase chromatogram condition in step (2):
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: filler is C
18, post footpath Φ 20cm or post footpath Φ 30cm,
Chromatographic column filler: 10 μm, 120A,
Moving phase: A phase 0.01 ~ 0.05% trifluoroacetic acid solution, B phase-acetonitrile,
Wavelength: 230nm,
Flow velocity: 800ml/ minute or 1600ml/ minute,
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, is raised to 35% from 20% in 60min in 1min; In 1min, be increased to 50%, 50% keeps 3min, in 1min, drop to 5%, keeps until wash-out terminates,
Circulation loading wash-out operates, and collects main peak solution.
And sample standard: main peak purity>=99.9%, known impurities ASP
9?Angiomax and D ?Phe
12?Angiomax all must not more than 0.1%.
By step (2) through the solution that the 2nd time purifying obtains, add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve and steam to steaming without acetonitrile, obtain Angiomax sterling solution, be distributed into dish.
Step (3) step of freeze drying:
Open cold lyophilizer, put into and divide the Angiomax sterling installed solution, freeze-drying is carried out :-45 DEG C of pre-freezes 3 ~ 5 hours by the freeze-drying curve of setting, be evacuated to 10 to 100Pa, trunk is dry to be warming up to 5 ~ 25 DEG C and to keep about 16 ~ 19 hours, and ice crystal is warming up to 25 DEG C and keeps about 3 ~ 5 hours after disappearing, then is warming up to 30 DEG C and keeps outlet after about 20 hours, obtain Angiomax bulk drug finished product, freeze-drying time amounts to needs 42 ~ 56 hours.
Preferably, purification process of the present invention, comprises the following steps:
(1) get Angiomax crude product, add purified water and dissolve and be mixed with crude product solution, subsequently crude product solution is injected high performance liquid chromatograph and carry out purifying, purification condition is:
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: C18, post footpath Φ 15cm,
Chromatographic column filler: 45 μm, 100A,
Mobile phase A: 0.1% hyptafluorobutyric acid solution, Mobile phase B: acetonitrile,
Wavelength: 230nm,
Flow velocity: 400ml/ minute,
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, keeps 60min isocratic elution in 1min; In 1min, be increased to 50%, 50% keeps 5min, in 1min, drop to 5%, keeps until wash-out terminates,
Sample size :≤50g/ pin,
Circulation loading wash-out operates, and collects main peak solution,
And sample standard: main peak purity >=99.6%,
To learn from else's experience the Angiomax solution of above-mentioned high performance liquid chromatograph purifying, added in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve steam to vacuum tightness Yue ? below 0.09Mpa, obtain the thick pure solution of Angiomax,
(2) the thick pure solution of the Angiomax upper step prepared, is injected into preparative high performance liquid chromatography instrument and carries out second time purifying, purification condition:
The applied sample amount 50g/ pin of the thick pure solution of Angiomax,
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: C18, post footpath Φ 20cm or post footpath Φ 30cm,
Chromatographic column filler: 10 μm, 120A,
Moving phase: A phase: 0.025% trifluoroacetic acid solution, B phase: acetonitrile,
Wavelength: 230nm,
Flow velocity: for Φ 20cm chromatographic column, 800ml/ minute; For Φ 30cm chromatographic column, 1600ml/ minute,
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, is raised to 35% from 20% in 60min in 1min; In 1min, be increased to 50%, 50% keeps 3min, in 1min, drop to 5%, keeps until wash-out terminates,
Circulation loading wash-out operates, and collects main peak solution,
And sample standard: main peak purity >=99.9%,
By the sample solution of above-mentioned consummate main peak purity >=99.9% obtained, add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve and steam to vacuum tightness about below-0.09Mpa, obtain Angiomax sterling solution, be distributed into dish
(3) open cold lyophilizer, put into and divide the Angiomax sterling installed solution, freeze-drying is carried out :-45 DEG C of pre-freezes 3 hours by the freeze-drying curve of setting, be evacuated to 10 to 100Pa, trunk is dry to be warming up to 25 DEG C and to keep about 19 hours, and ice crystal is warming up to 25 DEG C and keeps about 5 hours after disappearing, then is warming up to 30 DEG C and keeps outlet after about 20 hours, obtain Angiomax bulk drug finished product, described freeze-drying time amounts to and about needs 56 hours.
Angiomax crude product of the present invention belongs to currently available products, can commercially buy.
Raw material of the present invention is provided by Hainan Zhonghe Pharmaceutical Co., Ltd.
Angiomax HPLC method for detecting purity: according to high performance liquid chromatography < Chinese Pharmacopoeia version in 2010 two annex V 0) measure.
Chromatographic condition and system suitability
Chromatographic column: Agilent Extend-C
18post (250 × 4.6m 5 μm), with 0.1mol/L sodium phosphate buffer (14.2g anhydrous sodium sulphate, the about 800ml that adds water dissolves, add phosphoric acid 5.4ml, by triethylamine adjust ph to 7.0, be settled to 100ml) be mobile phase A, with water: second dried meat (10:90) is Mobile phase B; Flow velocity is 1.0ml/min; Column temperature 25 DEG C; Determined wavelength is 214nm; Number of theoretical plate should be not less than 3000 by Angiomax, and Angiomax peak and the high and steep resolution of impurity should conform with the regulations.By following gradient:
Get this product appropriate, accurately weighed, by water dissolution and the solution be diluted to containing 0.5mg Angiomax in 1ml, shake up, as need testing solution; Precision measures trial-product liquation 50 μ L injection liquid chromatography, and record need testing solution color atlas to 2 times of principal constituent peak retention time calculate main peak purity according to area normalization method.
The purification process of Angiomax of the present invention, be through screening to obtain: for Angiomax crude product, twice gradient elution is adopted to carry out purifying, first time, purifying adopted hyptafluorobutyric acid system (gradient comprising specific moving phase composition and optimize), and this purification process can effectively remove the impurity produced in most of synthesis technique after using; Second time adopts trifluoroacetic acid system (gradient comprising specific moving phase composition and optimize), second time purifying disposable realization can reduce the foreign matter content (purity can reach 99.90%) of the solution of first time purifying and completes and turn salt process, and Angiomax fine work solution rear impurity after the freeze-dry process freeze-drying after optimizing of acquisition does not change.
Compared with the conventional method comparatively, operation sequence is simple in the present invention, effectively improves purity and the yield of Angiomax, controls foreign matter content.The purity of Angiomax of the present invention more than 99.90%, impurity A SP
9?Angiomax and D ?Phe
12?Angiomax must not more than 0.5%.After placing through long-time (more than 2 years), its active constituent content content, shows extraordinary stability.Meanwhile, purifying process of the present invention is simple to operate, and cost is low, is applicable to scale operation.The drug quality prepared by the present invention is more reliable, and security is higher.
Accompanying drawing illustrates:
Fig. 1: the related substance detected result of Angiomax crude product
Fig. 2: the related substance detected result of the main peak solution that this product is collected after step one purifying
Fig. 3: the related substance detected result of main peak solution solution after concentrated that this product is collected after step 2 purifying
Fig. 4: the related substance detected result of this product Angiomax bulk drug after step 3 freeze-drying
Embodiment:
By following specific embodiment, the present invention is further illustrated, but not as restriction of the present invention.
Embodiment 1, purification process of the present invention
Step one: Angiomax crude product slightly pure: Angiomax crude product (Hainan Zhonghe Pharmaceutical Co., Ltd provides, HPLC purity 87.47%, accompanying drawing 1) adds purified water and dissolves and the sample crude product solution being mixed with respective concentration.
Instrument: preparative high performance liquid chromatography instrument
Chromatographic column: C18, post footpath Φ 15cm
Chromatographic column filler: 45 μm, 100A
Mobile phase A: 0.1% hyptafluorobutyric acid solution, Mobile phase B: acetonitrile (chromatographically pure)
Wavelength: 230nm
Flow velocity: 400ml/ minute
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, keeps 60min isocratic elution in 1min; In 1min, be increased to 50%, 50% keeps 5min, in 1min, drop to 5%, keeps until wash-out terminates.
Sample size :≤50g/ pin
Circulation loading wash-out operates, and collects main peak solution.
And sample standard: main peak purity >=99.6%.
Get the thick pure solution of above-mentioned Angiomax, add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve steam to vacuum tightness Yue ? below 0.09Mpa (steaming without acetonitrile), obtain the thick pure solution of Angiomax (HPLC purity 99.83%, accompanying drawing 2).
Step 2: Angiomax consummate
Injecting preparative high performance liquid chromatography instrument by following condition carries out consummate.The applied sample amount 50g/ pin of the thick pure solution of Angiomax.
Instrument: preparative high performance liquid chromatography instrument
Chromatographic column: C18, post footpath Φ 20cm or post footpath Φ 30cm
Chromatographic column filler: 10 μm, 120A
Moving phase: A phase 0.025% trifluoroacetic acid solution, B phase-acetonitrile (chromatographically pure)
Wavelength: 230nm
Flow velocity: 800ml/ minute (Φ 20cm chromatographic column) or 1600ml/ minute (Φ 30cm chromatographic column)
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, is raised to 35% from 20% in 60min in 1min; In 1min, be increased to 50%, 50% keeps 3min, in 1min, drop to 5%, keeps until wash-out terminates.
Circulation loading wash-out operates, and collects main peak solution.
And sample standard: main peak purity >=99.9%.
By the sample solution of above-mentioned consummate main peak purity >=99.9% obtained, add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve and steam to vacuum tightness about below-0.09Mpa (steaming without acetonitrile), obtain Angiomax sterling solution (HPLC purity 99.91%, accompanying drawing 3), be distributed into dish.
Step 3: freeze-drying
Open cold lyophilizer, put into and divide the Angiomax sterling installed solution, freeze-drying is carried out :-45 DEG C of pre-freezes 3 hours by the freeze-drying curve of setting, be evacuated to 10 to 100Pa, trunk is dry is warming up to 25 DEG C of maintenances about 19 hours, note the ice crystal Disappearance Scenarios observing goods, the ice crystal Disappearance Scenarios according to goods can suitably shorten or extend this phases-time; Ice crystal is warming up to 25 DEG C and keeps about 5 hours after disappearing, then is warming up to 30 DEG C and keeps outlet after about 20 hours, obtains Angiomax bulk drug finished product.Freeze-drying time amounts to and about needs 56 hours.Measure Angiomax bulk drug HPLC purity 99.90% (accompanying drawing 4).
Claims (10)
1. a purifying process for Bivalirudin, is characterized in that, comprises the following steps:
(1) Bivalirudin crude product is got, add purified water dissolve and be mixed with crude product solution, subsequently crude product solution is injected high performance liquid chromatograph and carry out purifying, purification condition is: chromatographic is by mobile phase A: 0.025 to 0.2% hyptafluorobutyric acid solution and Mobile phase B form, chromatographic column filler is the octadecylsilane chemically bonded silica filler of particle diameter 45 μm ~ 75 μm, after gradient elution, purity can reach 99.8%
(2) the thick pure solution of the Bivalirudin upper step prepared, be injected into preparative high performance liquid chromatography instrument and carry out second time purifying, purification condition: chromatographic is by mobile phase A: 0.01 ~ 0.05% trifluoroacetic acid solution and Mobile phase B form, chromatographic column filler is the octadecylsilane chemically bonded silica filler of particle diameter 10 μm, gradient elution, solution after twice purifying concentrates, and the purity of concentrated solution can reach 99.90%
(3) concentrated solution freeze-drying, to obtain final product.
2. purifying process according to claim 1, is characterized in that, high-efficient liquid phase chromatogram condition in step (1):
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: C
18, post footpath Φ 15cm,
Chromatographic column filler: 45 μm ~ 75 μm, 100A
Mobile phase A: 0.025 to 0.2% hyptafluorobutyric acid solution, Mobile phase B: acetonitrile,
Wavelength: 230nm,
Flow velocity: 400ml/ minute,
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, keeps 60min isocratic elution in 1min; In 1min, be increased to 50%, 50% keeps 5min, in 1min, drop to 5%, keeps until wash-out terminates,
Sample size :≤50g/ pin
Circulation loading wash-out operates, and collects main peak solution.
3. purifying process according to claim 1, is characterized in that, high-efficient liquid phase chromatogram condition in step (1): and sample standard: main peak purity >=99.6%.
4. purifying process according to claim 1, it is characterized in that, get the solution of the Bivalirudin obtained through purifying in step (1), add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve steam to vacuum tightness Yue ? below 0.09Mpa, obtain the thick pure solution of Bivalirudin.
5. purifying process according to claim 1, is characterized in that, high-efficient liquid phase chromatogram condition in step (2):
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: filler is C
18, post footpath Φ 20cm or post footpath Φ 30cm,
Chromatographic column filler: 10 μm, 120A,
Moving phase: A phase 0.01 ~ 0.05% trifluoroacetic acid solution, B phase-acetonitrile,
Wavelength: 230nm,
Flow velocity: 800ml/ minute or 1600ml/ minute,
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, is raised to 35% from 20% in 60min in 1min; In 1min, be increased to 50%, 50% keeps 3min, in 1min, drop to 5%, keeps until wash-out terminates,
Circulation loading wash-out operates, and collects main peak solution.
6. purifying process according to claim 1, is characterized in that, high-efficient liquid phase chromatogram condition in step (2): and sample standard: main peak purity>=99.9%, known impurities ASP
9?Angiomax and D ?Phe
12?Angiomax all must not more than 0.1%.
7. purifying process according to claim 1, it is characterized in that, by step (2) through solution that the 2nd time purifying obtains, add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve and steam to steaming without acetonitrile, obtain Bivalirudin sterling solution, be distributed into dish.
8. purifying process according to claim 1, it is characterized in that, step (3) step of freeze drying: open cold lyophilizer, put into and divide the Bivalirudin sterling installed solution, freeze-drying is carried out :-45 DEG C of pre-freezes 3 ~ 5 hours by the freeze-drying curve of setting, be evacuated to 10 to 100Pa, trunk is dry is warming up to 5 ~ 25 DEG C of maintenances about 16 ~ 19 hours, ice crystal is warming up to 25 DEG C and keeps about 3 ~ 5 hours after disappearing, be warming up to 30 DEG C again and keep outlet after about 20 hours, obtain Bivalirudin bulk drug finished product, freeze-drying time amounts to needs 42 ~ 56 hours.
9. purifying process according to claim 1, is characterized in that, comprises the following steps:
(1) get Bivalirudin crude product, add purified water and dissolve and be mixed with crude product solution, subsequently crude product solution is injected high performance liquid chromatograph and carry out purifying, purification condition is:
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: C18, post footpath Φ 15cm,
Chromatographic column filler: 45 μm, 100A,
Mobile phase A: 0.1% hyptafluorobutyric acid solution, Mobile phase B: acetonitrile,
Wavelength: 230nm,
Flow velocity: 400ml/ minute,
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, keeps 60min isocratic elution in 1min; In 1min, be increased to 50%, 50% keeps 5min, in 1min, drop to 5%, keeps until wash-out terminates,
Sample size :≤50g/ pin,
Circulation loading wash-out operates, and collects main peak solution,
And sample standard: main peak purity >=99.6%,
To learn from else's experience the Bivalirudin solution of above-mentioned high performance liquid chromatograph purifying, added in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve steam to vacuum tightness Yue ? below 0.09Mpa, obtain the thick pure solution of Bivalirudin,
(2) the thick pure solution of the Bivalirudin upper step prepared, is injected into preparative high performance liquid chromatography instrument and carries out second time purifying, purification condition:
The applied sample amount 50g/ pin of the thick pure solution of Bivalirudin,
Instrument: preparative high performance liquid chromatography instrument,
Chromatographic column: C18, post footpath Φ 20cm or post footpath Φ 30cm,
Chromatographic column filler: 10 μm, 120A,
Moving phase: A phase: 0.025% trifluoroacetic acid solution, B phase: acetonitrile,
Wavelength: 230nm,
Flow velocity: for Φ 20cm chromatographic column, 800ml/ minute; For Φ 30cm chromatographic column, 1600ml/ minute,
Gradient: Mobile phase B 5% balance 15min, is raised to 20%, is raised to 35% from 20% in 60min in 1min; In 1min, be increased to 50%, 50% keeps 3min, in 1min, drop to 5%, keeps until wash-out terminates,
Circulation loading wash-out operates, and collects main peak solution,
And sample standard: main peak purity >=99.9%,
By the sample solution of above-mentioned consummate main peak purity >=99.9% obtained, add in Rotary Evaporators, control temperature 25 ± 2 DEG C, in 0 DEG C ~ 10 DEG C circulating condensings, revolve and steam to vacuum tightness about below-0.09Mpa, obtain Bivalirudin sterling solution, be distributed into dish
(3) open cold lyophilizer, put into and divide the Bivalirudin sterling installed solution, freeze-drying is carried out :-45 DEG C of pre-freezes 3 hours by the freeze-drying curve of setting, be evacuated to 10 to 100Pa, trunk is dry is warming up to 25 DEG C of maintenances about 19 hours, ice crystal is warming up to 25 DEG C and keeps about 5 hours after disappearing, then is warming up to 30 DEG C and keeps outlet after about 20 hours, obtains Bivalirudin bulk drug finished product.
10. purifying process according to claim 9, is characterized in that, described freeze-drying time amounts to and about needs 56 hours.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106188218A (en) * | 2016-07-14 | 2016-12-07 | 江苏诺泰生物制药股份有限公司 | A kind of method improving polypeptide raw material drug stabilisation |
| CN113504330A (en) * | 2021-06-18 | 2021-10-15 | 安莱博医药(苏州)有限公司 | Process and device for purifying oligomeric dimethicone |
| CN117088966A (en) * | 2022-12-29 | 2023-11-21 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthesis method of bivalirudin impurity |
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| WO2011071799A2 (en) * | 2009-12-11 | 2011-06-16 | Dr. Reddy's Laboratories Ltd. | Purification of bivalirudin |
| CN102702325A (en) * | 2012-06-19 | 2012-10-03 | 深圳翰宇药业股份有限公司 | Preparation method of anticoagulant polypeptide |
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| WO2011071799A2 (en) * | 2009-12-11 | 2011-06-16 | Dr. Reddy's Laboratories Ltd. | Purification of bivalirudin |
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| CN106188218A (en) * | 2016-07-14 | 2016-12-07 | 江苏诺泰生物制药股份有限公司 | A kind of method improving polypeptide raw material drug stabilisation |
| CN113504330A (en) * | 2021-06-18 | 2021-10-15 | 安莱博医药(苏州)有限公司 | Process and device for purifying oligomeric dimethicone |
| CN117088966A (en) * | 2022-12-29 | 2023-11-21 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthesis method of bivalirudin impurity |
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