CN109721654A - A kind of isolation and purification method of Angiomax - Google Patents
A kind of isolation and purification method of Angiomax Download PDFInfo
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- CN109721654A CN109721654A CN201910199565.XA CN201910199565A CN109721654A CN 109721654 A CN109721654 A CN 109721654A CN 201910199565 A CN201910199565 A CN 201910199565A CN 109721654 A CN109721654 A CN 109721654A
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Abstract
The present invention provides a kind of isolation and purification methods of Angiomax.For the isolation and purification method of Angiomax of the invention the following steps are included: 1) dissolving Angiomax crude product, filtering obtains Angiomax solution；2) the Angiomax solution of step 1) preparing in column equipped with reverse phase filler is loaded to purify；It is eluted using acidity flowing relative target product, Fractional Collections purpose peak value solution, the solution for mixing each section of collection is to merge component liquid, obtains Angiomax after purification.The present invention is purified by a step, only needs a kind of filler that high-purity Angiomax can be obtained, and purification process is simple and easy to do, can be used for large-scale production, reduces production cost, improves production efficiency.
Description
Technical field
The invention belongs to technical field of medicine synthesis, are related to a kind of isolation and purification method more particularly to one of Angiomax
One step high efficiency separation and purification method of kind Angiomax.
Background technique
Angiomax (Bivalirudin, also referred to as bivalirudin) is a kind of direct thrombin inhibitor (DTI), is derived from
Hirudin derivative is a kind of polypeptide synthesized by 28 kinds of chemistry of amino acids, and 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。
Angiomax is a kind of effective, highly selective reversible inhibitor, can not only inhibit sequestered and mating type
Fibrin ferment, moreover it is possible to inhibit the platelet activation and aggregation by thrombin-mediated.The drug effect of Angiomax is fast, half-life short, in machine
In vivo not with plasma protein and erythrocyte binding, to heparin-induced thrombocytopenia and heparin-induced thrombosis-blood
Platelet reduces syndrome (HIT/HITTS) and risk is not present, in addition, do not need to combine the confactors such as antithrombase in treatment,
Without activation blood platelet, these features make Angiomax become an ideal heparin substitute.
The injectable dosage forms in December, 2000, the drug of Medicines company, U.S. exploitation list in the U.S., trade name
For Angiomax, general entitled Bivalirudin, the entitled Angiomax of Chinese, listing dosage form is freeze drying powder injection, and specification is
250mg/ branch.1~2 month 2005, Angiox (Bivalirudin of the Medicines company in the subsidiary Nycomed in Europe
Trade name in Europe) in Austria, Denmark.Finland, Germany, Switzerland and Britain's listing, intervene patient for percutaneous coronary artery,
The aseptic freeze-dried powder that its specification listed is 250mg/ bottles, Angiomax is for being injected intravenously and instiling.
Currently, preparing the method for Angiomax it has been reported that Solid-phase synthesis peptides and liquid phase synthesis can be used
Method prepares the thick peptide of Angiomax, and (according to substance detecting method related in national drug standards YBH02852011, impurity content is more than
Angiomax crude product specified in standard can be defined as the thick peptide of Angiomax), but it is directed to the purifying of the thick peptide of Angiomax, only
There is document report few in number.
CN101475631A discloses a kind of liquid-phase synthesis process of bivalirudin, and this method is merely depicted using HPLC
Method purifies Angiomax just peptide and the technique of freeze-drying, has no the description of detailed purifying process and lyophilized technique, and produce
Product purity is only capable of reaching 98.5%, must not exceed 1.5% rule according to related substance in national drug standards YBH02852011
Fixed, the Angiomax bulk pharmaceutical chemicals of preparation are already close to the limit of impurities, and within 2 years validity periods, long term storage easily causes related
Substance check item is unqualified.
CN102264757B discloses a kind of method for preparing bivalirudin, and the invention provides a kind of purifying Angiomaxes
The method of first peptide, purifies the crude peptide using preparation scale HPLC in C18 reverse phase stationary phase.The first step, using ammonium acetate/
Water/acetonihile gradient elution purifies the crude peptide, then second step, using trifluoroacetic acid/water/acetonihile gradient elution.But
The undeclared condition specifically purified of the invention, can not reappear its experimental result.
CN102286076A discloses a kind of preparation method of bivalirudin, and this invention describes the pure of Angiomax crude product
Change method: weighing Angiomax crude powder, and crude product is added into purified water (about 20mL water/g crude product), and it is dilute to stir lower dropwise addition
Ammonium hydroxide tune pH, by pH control 3.0~5.0,0.45 μm of mixing filtering with microporous membrane of solution, standby purifying is used.Using efficient liquid
Phase chromatography is purified, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, and flow phase system is 0.1%TFA/ aqueous solution-
The column flow rate of 0.1%TFA/ acetonitrile solution, 77mm × 250mm is 90mL/min, is eluted using gradient system, and sample introduction is recycled
Purifying, takes crude product solution to be splined in chromatographic column, and it is pure to obtain Angiomax after collection main peak boils off acetonitrile for starting mobile phase elution
Change intermediate concentrate.Angiomax is taken to purify intermediate concentrate, it is spare with 0.45 μm of filter membrane filtration.Using high-efficient liquid phase color
Spectrometry carries out changing salt, and flow phase system is 0.1%TFA/ aqueous solution-acetonitrile, and it is 10 μm of reverse phase C18 that chromatograph packing material is used in purifying,
The column flow rate of 77mm × 250mm is 90ml/min (can adjust corresponding flow velocity according to the chromatographic column of different size).Using
Gradient elution, quadrat method in circulation, is splined in chromatographic column, and starting mobile phase elution acquires map, observes the change of trap
Change, collection changes salt main peak and detects purity with analysis liquid phase, and merging is changed salt main peak solution, depressurized under less than 40 DEG C water bath conditions
Concentration, boils off most of acetonitrile with Rotary Evaporators, obtains Angiomax trifluoroacetic acid aqueous solution, is freeze-dried, obtains product
608g, total recovery 55.8%.But only with a kind of chromatogram purification system in the invention, and gradient elution is not announced
Specific embodiment is needed by collecting the Angiomax met in national drug standards YBH02852011 in relation to substance repeatedly
Solution is concentrated after merging, freeze-drying.This method will cause Angiomax bulk pharmaceutical chemicals in purification process, because only that a kind of flowing
Phase composition is eluted, and the separating degree of impurity and Angiomax is unable to ensure, and needs repeatedly to collect the higher product of purity repeatedly
Solution, loss greatly, are unfavorable for the bulk pharmaceutical chemicals of preparation of industrialization feather weight.
CN103965293A discloses a kind of high-purity Angiomax and its industrialized process for preparing: gained crude product is used
Reversed-phase high performance liquid chromatography gradient elution purifies and separates, using octadecylsilane chemically bonded silica as stationary phase, eluant, eluent w (TFA):
V (acetonitrile): V (water)=1:250:750~1:400:600, flow velocity 500mL/min, Detection wavelength 214nm.What revolving was collected into
Eluent obtains Angiomax 0.54kg, purity 99.8%, gross production rate 37% after freeze-drying.The method of the invention is equally only
Using a kind of chromatogram purification system, gradient has no detailed description, can not reappear product preparation process.
CN103242431A discloses a kind of preparation method of bivalirudin, and which depict the purifying sides of Angiomax crude product
Method: being weighed the Angiomax crude powder 8.0g of preparation, dissolved using acetonitrile solution, 0.45 μm of miillpore filter mistake of solution
Filter, it is spare.Condition when high performance liquid chromatography is purified, chromatographic column: the octadecylsilane chemically bonded silica with 10 μm is
Stationary phase, pillar diameter and length are as follows: 50mm × 250mm；Mobile phase: 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solution；
The flow velocity 60mL/min of elution；Using gradient elution, input mode loading is recycled.The sample solution of above-mentioned processing is splined on color
It composes in column, starting mobile phase elution, collect main peak and detects purity with analysis liquid phase, merge main peak solution, in less than 40 DEG C water
It is concentrated under reduced pressure under the conditions of bath, boils off most of acetonitrile with Rotary Evaporators, obtain Angiomax trifluoroacetic acid salting liquid.Freeze-drying
Obtain product 2.48g, total recovery 31%, product purity 99.6%, Angiomax [+Gly] impurity, Angiomax [- Gly] impurity
Respectively less than 0.1%, obtain Angiomax sterling.The method of the invention is equally only with a kind of chromatogram purification system, and gradient is not
See detailed description, and preparative-scale is only 2.5g, it is on the knees of the gods for the sample feasibility for producing feather weight.
CN103319570A discloses a kind of preparation method of bivalirudin, and embodiment 12 discloses Angiomax fine peptide
The preparation method of trifluoroacetate: weigh 200g according to the thick peptide of Angiomax obtained described in embodiment nine with 25% acetonitrile+
After the mixed solvent 5000mL dissolution of 25%DMSO+50% water, using Waters2545RP-HPLC system, wavelength 214nm, color
Spectrum column is 50 × 250mm reverse phase C8 column, and column temperature is 50 DEG C, and conventional 0.1%TFA/ acetonitrile mobile phase purifying is collected purpose peak and evaporated
Point, purity is obtained greater than 98.5% fine peptide.By fine peptide solution use Waters2545RP-HPLC system, chromatographic column be 50 ×
250mm reverse phase C8 column, 0.1% trifluoracetic acid/acetonitrile mobile phase turn salt, collect purpose peak fraction, concentrated by rotary evaporation, and freeze-drying is compared
Lu Ding trifluoroacetate fine peptide 79.8g, HPLC purity 99.53% is cut down, yield 81.4%, total recovery 35.9% are purified.This method
Only with a kind of chromatogram purification system, gradient has no detailed description, and thick peptide dissolution has used 25% in purification process
DMSO (dimethyl sulfoxide) solution, and for preparing the bulk pharmaceutical chemicals that injection uses, in the quality control system of bulk pharmaceutical chemicals
The control for needing to carry out residual solvent for DMSO, increases cost and the period of detection, the HPLC purity 99.53% of product,
It must not exceed 1.5% according to substance related in national drug standards YBH02852011 is always miscellaneous, it is known that impurity A SP9- Angiomax
It must not exceed 0.5% regulation with D-Phe12- Angiomax, the Angiomax bulk pharmaceutical chemicals of the preparation are limited already close to total impurities
The 1/3 of degree, within 2 years validity periods, long term storage easily causes Related substances separation item unqualified and other issues.
The purifying process of the bulk pharmaceutical chemicals of above-mentioned Angiomax, product yield, production capacity are all relatively low or purity is relatively low, freeze-drying
Technique is unable to ensure easily during sample freeze-drying that known impurities ASP9- Angiomax does not increase, therefore is unfavorable for industrialized production
Feather weight, can ensure the Angiomax bulk pharmaceutical chemicals of Related substances separation item qualification before the deadline.
Therefore, for a series of problems existing for existing Angiomax bulk pharmaceutical chemicals purifying process, the present invention provides a kind of ratio
Cut down the isolation and purification method of Lu Ding.
Summary of the invention
In view of the deficiencies of the prior art, pure the purpose of the present invention is to provide a kind of isolation and purification method of Angiomax
Degree height, high income and stabilization, operation is simple, and stationary phase used is reusable, greatly reduces cost, improves production
Efficiency.
To achieve this purpose, the present invention adopts the following technical scheme:
A kind of isolation and purification method of Angiomax, the isolation and purification method the following steps are included:
1) Angiomax crude product is dissolved, filters, obtains Angiomax solution；
2) the Angiomax solution of step 1) preparing in column equipped with reverse phase filler is loaded to purify；Using acidity
Mobile phase elutes target product, Fractional Collections purpose peak value solution, and the solution for mixing each section of collection is to merge component liquid,
Obtain Angiomax after purification.
It is high-purity can to meet 99% or more Angiomax by step purifying for the isolation and purification method of Angiomax of the present invention
The quality requirement of degree, purification process are simple and easy to do；Separation method of the invention simultaneously only needs a kind of filler, can be used for scale metaplasia
It produces, greatly reduces production cost, improve production efficiency.
In step 2), the acidity mobile phase includes A phase and B phase；Preferably, the water of the A Xiang Weihan acid regulator
Solution, the organic solvent of the B Xiang Weihan acid regulator.
Preferably, in A phase, the volumetric concentration of the acid regulator is 0.03%~0.3%, such as acid regulator
Volumetric concentration 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%,
0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%,
0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29% or 0.3% etc..
Preferably, in B phase, the volumetric concentration of the acid regulator is 0.03%~0.3%, such as acid regulator
Volumetric concentration 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%,
0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%,
0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29% or 0.3% etc..
Wherein, the pH value of the acid mobile phase is 2.6~3.2, for example, the pH value of acid mobile phase is 2.6,2.7,
2.8、2.9、3、3.1、3.2。
The acid regulator is the mixture of any one or at least two in formic acid, phosphoric acid, hydrochloric acid and perchloric acid,
Preferably formic acid.The mixture typical case but unrestricted group are combined into the mixture of two kinds of acid regulators, such as formic acid and phosphorus
The mixture of acid, the mixture of formic acid and hydrochloric acid, the mixture of formic acid and perchloric acid, the mixture of phosphoric acid, hydrochloric acid, phosphoric acid and height
The mixture of the mixture of chloric acid, hydrochloric acid and perchloric acid；The mixture can also be the mixture of three kinds of acid regulators, example
As formic acid, phosphoric acid, hydrochloric acid mixture, the mixture of formic acid, phosphoric acid and perchloric acid, the mixture of phosphoric acid, hydrochloric acid and perchloric acid；
The mixture can also be four kinds of acid regulators mixture, such as formic acid, phosphoric acid, hydrochloric acid and perchloric acid mixture.
The organic solvent is the mixing of any one or at least two in acetonitrile, methanol, ethyl alcohol, acetone and isopropanol
Object, preferably acetonitrile.The mixture typical case but unrestricted group are combined into the mixture of two kinds of organic solvents, such as acetonitrile, first
The mixture of the mixture of the mixture of the mixture of alcohol, acetonitrile and ethyl alcohol, acetonitrile and acetone, acetonitrile and isopropanol；It is described mixed
Close the mixture that object can also be three kinds of organic solvents, such as the mixture of acetonitrile, methanol and ethyl alcohol, acetonitrile, methanol and acetone
Mixture, the mixture of acetonitrile, methanol and isopropanol, the mixture of acetonitrile, ethyl alcohol and acetone, acetonitrile, ethyl alcohol and isopropanol
Mixture, the mixture of acetonitrile, acetone and isopropanol, the mixture of methanol, ethyl alcohol and acetone, methanol, ethyl alcohol and isopropanol
Mixture, methanol, acetone, isopropanol mixture, the mixture of ethyl alcohol, acetone and isopropanol；The mixture can be with
For the mixture of four kinds of organic solvents, for example, acetonitrile, methanol, ethyl alcohol and acetone mixture, acetonitrile, methanol, ethyl alcohol and isopropyl
The mixture of alcohol, acetonitrile, ethyl alcohol, acetone and isopropanol mixture, methanol, ethyl alcohol, acetone and isopropanol mixture；It is described
Mixture may be the mixture of five kinds of organic solvents, for example, acetonitrile, methanol, ethyl alcohol, acetone and isopropanol mixture.
The present invention, by optimizing above-mentioned acid regulator and organic solvent and its volumetric concentration, so that the effect isolated and purified
Fruit is optimal.
In step 2), the model of the reverse phase filler is UniSil microballoon series；Preferably, the reverse phase filler is
UniSil Biva microballoon series.UniSil Biva microballoon is fitted for the uniform particle diameter of Angiomax separation special designing, aperture
Preferably, the good polymer microsphere of tolerance, the microballoon have the particle size and aperture structure of strict control, are used for it than cutting down
There is good specific aim when Lu Ding is isolated and purified.
In step 1), the purity of the Angiomax crude product is 60% or more.
In step 2), before the loading, further include the steps that preparing column equilibration；
Preferably, the detailed process of the balance are as follows: use acid mobile phase by A phase to column is prepared: B phase volume ratio for
(90~95): (5~10) are balanced, such as A phase in acid mobile phase: B phase volume ratio is 90:10,91:9,92:8,93:7,
94:6,95:5 etc..
In step 2), the elution is gradient elution；
Preferably, the detailed process of the gradient elution are as follows: the acid flowing for being first 5%~10% with B phase volume concentration
Mutually rinse 2 column volumes, after in 1 column volume with B phase volume concentration be 15~20% acid mobile phase rinse, then exist
It is rinsed in 10 column volumes with the acid mobile phase that B phase volume concentration is 25~30%.
It is highly preferred that the detailed process of the gradient 5%-28% elution are as follows: first rinse 2 with the B phase of volumetric concentration 5%
Column volume, after in 1 column volume B phase volume concentration gradient improved by 5% to 18%, the then B phase body in 10 column volumes
Product concentration gradient is improved by 18% to 28%.
As a preferred solution of the present invention, the isolation and purification method of the Angiomax includes the following steps:
1) the Angiomax crude product that purity is 60% or more is dissolved, filtering obtains Angiomax solution；
2) the Angiomax solution of step 1) is loaded to preparing equipped with UniSil microballoon to purify in column, using acid
Property mobile phase elutes target product, and the solution of Fractional Collections purpose peak value merges satisfactory component liquid；
Wherein, the acid mobile phase includes A phase formic acid-aqueous solution and B phase formic acid-acetonitrile solution；Fractional Collections purpose peak value it is molten
Liquid summarizes satisfactory component liquid, obtains Angiomax after purification.
Compared with prior art, the invention has the benefit that
(1) isolation and purification method of Angiomax of the invention, not only purity is high, high income, favorable reproducibility, but also operation
It is simple and easy, it is only necessary to step purifying, the purity of Angiomax after purification 99.76% or more, largest single impurity 0.15% with
Under, yield is 74% or more.
(2) isolation and purification method of Angiomax of the invention, using monodispersed UniSil microballoon as stationary phase, institute
It is reusable with stationary phase, cost is greatly reduced, production efficiency is improved.
Detailed description of the invention
Fig. 1 is the efficient liquid phase chromatographic analysis map of the Angiomax of the embodiment of the present invention 1 before purification；
Fig. 2 is the efficient liquid phase chromatographic analysis map of the Angiomax of the embodiment of the present invention 1 after purification；
Fig. 3 is the efficient liquid phase chromatographic analysis map of the Angiomax of the embodiment of the present invention 2 after purification.
Specific embodiment
1-3 with reference to the accompanying drawing, and pass through specific embodiment to further illustrate the technical scheme of the present invention.
Unless specific instructions, various raw materials of the invention are commercially available buys, or is prepared according to the conventional method of this field
It obtains.
Embodiment 1
The isolation and purification method of the Angiomax of the present embodiment, comprising the following steps:
Taking purity is 62.96% Angiomax coarse powder, and pure water dissolution, shaking ultrasound, concentration of ordinary dissolution 10mg/ is added
mL.After solution clarification, it is stand-by to collect filtrate for the membrane filtration for being 0.45 μm with aperture.Using the chromatographic column of 4.6 × 250mm,
UniSil Biva microballoon (production of Suzhou Na Wei Science and Technology Co., Ltd.) is used as and prepares filler, packed column volume 4.15mL.It is right
Prepare the acid mobile phase (A phase: 0.05% formic acid-aqueous solution, B phase: 0.05% formic acid-acetonitrile solution) of column with 5% B phase into
Row balance.After quantitative loop loading, gradient elution is carried out, flow control is in 0.6mL/min.Gradient elution process is first to use
5% B phase rinses 2 column volumes, after improved by 5% B to 18% B, then in 10 cylinders in 1 column volume inside gradient
Product inside gradient is improved by 18%B to 28%B, the solution of the Fractional Collections gradient section purpose peak value, to satisfactory component liquid
It merges, through efficient liquid phase chromatographic analysis, the purity of Angiomax is greater than 99.7% in amalgamation liquid, and yield is greater than 75%.
The efficient liquid phase chromatographic analysis map of Angiomax crude product before purification is pure as shown in Figure 1, as seen from Figure 1
There is a large amount of impurity in Angiomax before change.Fig. 2 is the Angiomax amalgamation liquid efficient liquid phase chromatographic analysis of a step after purification
Map, as seen from Figure 2, through a step, impurity content is extremely low after purification, and miscellaneous peak quantity is considerably less.
Embodiment 2
The isolation and purification method of the Angiomax of the present embodiment, comprising the following steps:
Taking purity is 62.96% Angiomax coarse powder, and pure water dissolution, shaking ultrasound, concentration of ordinary dissolution 10mg/ is added
mL.After solution clarification, it is stand-by to collect filtrate for the membrane filtration for being 0.45 μm with aperture.Using the chromatographic column of 4.6 × 250mm,
UniSil Biva microballoon (production of Suzhou Na Wei Science and Technology Co., Ltd.) is used as and prepares filler, packed column volume 4.15mL.It is right
The acid mobile phase (A phase: 0.1% formic acid-aqueous solution, B phase: 0.1% formic acid-acetonitrile solution) of column is prepared to carry out with 5% B phase
Balance.After quantitative loop loading, gradient elution is carried out, flow control is in 0.6mL/min.Gradient elution process is, first with 5%
B phase rinse 2 column volumes, after improved by 5% B to 18% B, then in 10 column volumes in 1 column volume inside gradient
Inside gradient is improved by 18%B to 28%B, the solution of the Fractional Collections gradient section purpose peak value, to satisfactory component liquid into
Row merges, and through efficient liquid phase chromatographic analysis, the purity of Angiomax is greater than 99.6% in amalgamation liquid, and yield is greater than 70%.
The efficient liquid phase chromatographic analysis map of Angiomax crude product before purification is pure as shown in Figure 1, as seen from Figure 1
There is a large amount of impurity in Angiomax before change.Fig. 3 is the Angiomax amalgamation liquid efficient liquid phase chromatographic analysis of a step after purification
Map, as seen from Figure 3, through a step, impurity content is extremely low after purification, and miscellaneous peak quantity is considerably less.
Comparative example 1
This comparative example difference from example 1 is that, it is different to purify mobile phase used.
Comparative example 1.1
The isolation and purification method of the Angiomax of this comparative example is as follows:
Taking purity is 62.96% Angiomax coarse powder, and pure water dissolution, shaking ultrasound, concentration of ordinary dissolution 10mg/ is added
mL.After solution clarification, it is stand-by to collect filtrate for the membrane filtration for being 0.45 μm with aperture.Using the chromatographic column of 4.6 × 250mm,
UniSil Biva microballoon (production of Suzhou Na Wei Science and Technology Co., Ltd.) is used as and prepares filler, packed column volume 4.15mL.It is right
The acid mobile phase (A phase: 0.05%TFA- aqueous solution, B phase: 0.05%TFA- acetonitrile solution) of column is prepared to carry out with 5% B phase
Balance.After quantitative loop loading, gradient elution is carried out, flow control is in 0.6mL/min.Gradient elution process is, first with 5%
B phase rinse 2 column volumes, after improved by 5% B to 18% B, then in 10 column volumes in 1 column volume inside gradient
Inside gradient is improved by 18%B to 28%B, the solution of the Fractional Collections gradient section purpose peak value, to satisfactory component liquid into
Row merges, and through efficient liquid phase chromatographic analysis, the purity of Angiomax is greater than 99.1% in amalgamation liquid, and yield is greater than 55%.
Comparative example 1.2
The isolation and purification method of the Angiomax of this comparative example is as follows:
Taking purity is 62.96% Angiomax coarse powder, and pure water dissolution, shaking ultrasound, concentration of ordinary dissolution 10mg/ is added
mL.After solution clarification, it is stand-by to collect filtrate for the membrane filtration for being 0.45 μm with aperture.Using the chromatographic column of 4.6 × 250mm,
UniSil Biva microballoon (production of Suzhou Na Wei Science and Technology Co., Ltd.) is used as and prepares filler, packed column volume 4.15ml.It is right
Prepare the acid mobile phase (A phase: 0.05% acetic acid-aqueous solution, B phase: 0.05% acetic acidacetonitrile solution) of column with 5% B phase into
Row balance.After quantitative loop loading, gradient elution is carried out, flow control is in 0.6mL/min.Gradient elution process is first to use
5% B phase rinses 2 column volumes, after improved by 5% B to 20% B, then in 10 cylinders in 1 column volume inside gradient
Product inside gradient is improved by 20%B to 30%B, the solution of the Fractional Collections gradient section purpose peak value, to satisfactory component liquid
It merges, through efficient liquid phase chromatographic analysis, the purity of Angiomax is greater than 99.2% in amalgamation liquid, and yield is greater than 49.78%.
Comparative example 1.3
The isolation and purification method of the Angiomax of this comparative example is as follows:
Taking purity is 62.96% Angiomax coarse powder, and pure water dissolution, shaking ultrasound, concentration of ordinary dissolution 10mg/ is added
mL.After solution clarification, it is stand-by to collect filtrate for the membrane filtration for being 0.45 μm with aperture.Using the chromatographic column of 4.6 × 250mm,
UniSil Biva microballoon (production of Suzhou Na Wei Science and Technology Co., Ltd.) is used as and prepares filler, packed column volume 4.15ml.It is right
The acid mobile phase (A phase: 0.05% acetic acid-aqueous solution, B phase: 0.05% acetic acid-aqueous isopropanol) of column is prepared with 5% B phase
It is balanced.After quantitative loop loading, gradient elution is carried out, flow control is in 0.6mL/min.Gradient elution process is, first
Rinse 2 column volumes with 5% B phase, after improved by 5% B to 20% B, then in 10 columns in 1 column volume inside gradient
Volume inside gradient is improved by 20%B to 30%B, the solution of the Fractional Collections gradient section purpose peak value, to satisfactory component
Liquid merges, and through efficient liquid phase chromatographic analysis, the purity of Angiomax is greater than 99.5% in amalgamation liquid, and yield is greater than 62%.
Comparative example 2
This comparative example difference from example 1 is that, it is different to purify stationary phase used.
The isolation and purification method of the Angiomax of this comparative example is as follows:
Taking purity is 62.96% Angiomax coarse powder, and pure water dissolution, shaking ultrasound, concentration of ordinary dissolution 10mg/ is added
mL.After solution clarification, it is stand-by to collect filtrate for the membrane filtration for being 0.45 μm with aperture.Using the chromatographic column of 4.6 × 250mm,
10-100C18 microballoon is used as and prepares filler, packed column volume 4.15ml.To preparing acid mobile phase (the A phase: 0.05% first of column
Acid-aqueous solution, B phase: 0.05% formic acid-acetonitrile solution) it is balanced with 5% B phase.After quantitative loop loading, ladder is carried out
Degree elution, flow control is in 0.6mL/min.Gradient elution process is first to rinse 2 column volumes with 5% B phase, after in 1 column
Volume inside gradient is improved by 5% B to 18% B, is then improved by 18%B to 28%B, segmentation in 10 column volume inside gradients
The solution for collecting the gradient section purpose peak value, merges satisfactory component liquid, through efficient liquid phase chromatographic analysis, merges
The purity of Angiomax is greater than 99.3% in liquid, and yield is greater than 55%.
In conclusion the experimental result of embodiment and comparative example is as shown in table 1:
Table 1
| Purity (%) | Largest single impurity (%) | Yield (%) | |
| Embodiment 1 | 99.797 | 0.143 | 77.904 |
| Embodiment 2 | 99.62 | 0.145 | 70.05 |
| Comparative example 1.1 | 99.1 | 0.21 | 55.301 |
| Comparative example 1.2 | 99.263 | 0.48 | 49.78 |
| Comparative example 1.3 | 99.531 | 0.28 | 62.13 |
| Comparative example 2 | 99.301 | 0.42 | 55.61 |
Comparative example 3
This comparative example is the purification process of CN104877024A.
Comparative example 4
This comparative example is the purification process of CN108663439A.
Comparative example 5
This comparative example is the purification process of CN103965293A.
Comparative example 6
This comparative example is the purification process of CN103319570A.
The embodiment of the present invention 1 is with the obvious advantage compared with the purification process of comparative example 3-6 simultaneously, specific relatively such as 2 institute of table
Show:
Table 2
The present invention is used for the polishing purification of Angiomax, and the quality requirement of Angiomax can be met by step purifying,
Purification process is simple and easy to do, and the purity of Angiomax after purification is 99.76% or more, and largest single impurity is 0.15% hereinafter, receiving
Rate is 74% or more；Separation method of the invention simultaneously only needs a kind of filler, can be used for large-scale production, greatly reduces production
Cost improves production efficiency.
As can be seen from Table 1, after being increased using the concentration of mobile phase, purity, largest single impurity and the yield of product are all
Decline.
After comparative example 1 is changed using the type of mobile phase, purity, largest single impurity and the yield of product are all declined.
After the stationary phase that comparative example 2 uses becomes 10-100C18 microballoon, purity, largest single impurity and the yield of product have
Declined.
In comparative example 3, the purity of fine peptide obtained is 99.9%, but the purity is high of its thick peptide selected, and is
87.47%, and two-step purifying is used, first step purifying is purified in high performance liquid chromatograph, purification condition are as follows: chromatography body
System is by mobile phase A: 0.025 to 0.2% hyptafluorobutyric acid solution and Mobile phase B form, and chromatographic column filler is 45 μm~75 μm of partial size
Octadecylsilane chemically bonded silica filler, second purifying purified in high performance liquid chromatograph, purification condition are as follows: chromatography
System is by mobile phase A: 0.01~0.05% trifluoroacetic acid solution and Mobile phase B form, and chromatographic column filler is the ten of 10 μm of partial size
Eight alkyl silane bonded silica gel fillers, gradient elution are concentrated through solution twice after purification, take a long time, the usage amount of reagent
It is larger.
Comparative example 4 isolates and purifies filtered crude product with highly effective liquid phase chromatographic system, using C18 as stationary phase, fills out
5~75 μm of material granularity, aqueous acetic acid: acetonitrile=80:20 is that mobile phase is eluted, and the purity of fine peptide obtained is
95.2%, although also using step purifying, its product purity obtained is lower.
The reversed-phase HPLC method gradient elution of comparative example 5 purifies crude product, eluant, eluent w (TFA): V (acetonitrile): V (water)=
1:250:750~1:400:600 collects eluant, eluent, and sterling is obtained after dry, and the purity of fine peptide obtained is 99.8%, but its
The purity is high for the thick peptide selected is 86%, and still, the yield of product is low, is 28%.
The purifying of comparative example 6 is reversed-phase high performance liquid chromatography purifying, and the chromatographic column of reversed-phase high performance liquid chromatography is reverse phase C18
Column, the purity of fine peptide obtained are 99.53%, although also using step purifying, its product yield obtained is lower, is
35.9%, production capacity is relatively low.
The isolation and purification method of Angiomax of the invention is completed to purify using one-step method, and process is easy, high-efficient, at
This is low, and product purity, yield are all relatively high.Meanwhile technique of the invention is lower for crude product purity requirement, greatly reduces
Requirement to upstream product.
Above embodiments are only used to illustrate method detailed of the invention, and the invention is not limited to above-mentioned method detaileds, i.e.,
Do not mean that the invention must rely on the above detailed methods to implement.It should be clear to those skilled in the art, right
Any improvement of the invention, the addition of equivalence replacement and auxiliary element to each raw material of product of the present invention, the selection of concrete mode
Deng all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of isolation and purification method of Angiomax, which is characterized in that the isolation and purification method the following steps are included:
1) Angiomax crude product is dissolved, filters, obtains Angiomax solution；
2) the Angiomax solution of step 1) preparing in column equipped with reverse phase filler is loaded to purify；It is flowed using acidity
Relative target product is eluted, Fractional Collections purpose peak value solution, and the solution for mixing each section of collection is to merge component liquid, is obtained
Angiomax after purification.
2. isolation and purification method according to claim 1, which is characterized in that in step 2), the acidity mobile phase includes A
Phase and B phase；
Preferably, the aqueous solution of the A Xiang Weihan acid regulator, the organic solvent of the B Xiang Weihan acid regulator；
Preferably, in A phase, the volumetric concentration of the acid regulator is 0.03%~0.3%；
Preferably, in B phase, the volumetric concentration of the acid regulator is 0.03%~0.3%.
3. isolation and purification method according to claim 1 or 2, which is characterized in that the pH value of the acidity mobile phase is 2.6
~3.2.
4. isolation and purification method according to claim 2, which is characterized in that the acid regulator is formic acid, phosphoric acid, salt
In acid and perchloric acid any one or at least two mixture, preferably formic acid.
5. isolation and purification method according to claim 2, which is characterized in that the organic solvent is acetonitrile, methanol, second
In alcohol, acetone and isopropanol any one or at least two mixture, preferably acetonitrile.
6. isolation and purification method described in one of -5 according to claim 1, which is characterized in that in step 2), the reverse phase filler
Model be UniSil microballoon series；
Preferably, the reverse phase filler is UniSil Biva microballoon series.
7. isolation and purification method described in one of -6 according to claim 1, which is characterized in that in step 1), the Angiomax
The purity of crude product is 60% or more.
8. the isolation and purification method according to one of claim 2-7, which is characterized in that in step 2), before the loading, also
Include the steps that preparing column equilibration；
Preferably, the detailed process of the balance are as follows: use acid mobile phase by A phase to column is prepared: B phase volume ratio for (90~
95): (5~10) are balanced.
9. isolation and purification method described in one of -8 according to claim 1, which is characterized in that in step 2), the elution is ladder
Degree elution；
Preferably, the detailed process of the gradient elution are as follows: first rushed with the acid mobile phase that B phase volume concentration is 5%~10%
Wash 2 column volumes, after in 1 column volume with B phase volume concentration be 15%~20% acid mobile phase rinse, then 10
It is rinsed in a column volume with the acid mobile phase that B phase volume concentration is 25%~30%.
10. isolation and purification method described in one of -9 according to claim 1, which is characterized in that the isolation and purification method includes
Following steps:
1) the Angiomax crude product that purity is 60% or more is dissolved, filtering obtains Angiomax solution；
2) the Angiomax solution of step 1) is loaded to preparing equipped with UniSil microballoon to purify in column, using acid stream
Dynamic relative target product is eluted, and the solution of Fractional Collections purpose peak value merges satisfactory component liquid；Its
In, the acidity mobile phase includes A phase formic acid-aqueous solution and B phase formic acid-acetonitrile solution；The solution of Fractional Collections purpose peak value,
Satisfactory component liquid is summarized, Angiomax after purification is obtained.
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