CN102875664B - Purifying method of carperitide - Google Patents
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Abstract
The invention provides a purifying method of carperitide. The purifying method comprises the following steps of: 1) dissolving a sample through 3 to 18% of an aqueous solution of an organic solvent based on volume by volume concentration, wherein the organic solvent has high polarity; preparing to obtain 100g/L of solution based on concentration; 2), getting octadecylsilane bonded silica gel as a stationary phase, getting 10 to 50mmol/L of sulfuric acid buffer salt in which 0.1 to 0.8% of perchloric acid is added based on a volume ratio as phase A, and getting acetonitrile as phase B, wherein volume ratio concentration of the phase B is 17 to 37%; feeding a solution prepared in step 1) to carry out gradient elution; and 3) transferring the carperitide sulfate into hydrochloride; carrying out a reverse phase HPLC (High Performance Liquid Chromatography) method; getting the octadecylsilane bonded silica gel as the stationary phase; carrying out the gradient elution; and drying to obtain the carperitide in a freezing way. The purifying method of the carperitide provided by the invention is stable and controllable in technology, high in yield, high in purity, wide in practical value and wide in application prospect.
Description
Technical field
The present invention relates to a kind of purification process of polypeptide drugs, the particularly purification process of Carperitide, belongs to pharmaceutical chemistry field.
Background technology
Acute heart failure (comprising chronic heart failure acute exacerbation) heart failure refers to a kind for the treatment of that causes abnormal gene expression to cause due to energy shortage, eventually because of energy shortage, myocardial ischemia, myocardial cell's apoptosis, myocardial contraction unit reduces, and makes this excess load enter vicious cycle.
Carperitide, commodity card by name piperazine Li Ting, the one circulation that is 28 amino acid compositions regulates hormone, and its molecular formula is: H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met – Asp-Arg-IIe-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH.Carperitide is treated the acute heart failure polypeptide drugs of (comprising that chronic heart failure increases the weight of) as one, it is by stimulating cardiac muscle to stretch, synthesized by ventricle endoparticle, then by hat vein distribution whole body, the tissues such as vasoactive unstriated muscle and kidney, regulate blood pressure and cylinder electrolyte balance.Clinical trial proves, Carperitide in the tissue such as vascular smooth muscle and kidney with GC-A receptors bind, activation guanylate cyclase, increases cyclic guanosine monophosphate, and causes that vasodilation and diuresis use.
At present, less about the research report of Carperitide, patent number is few both at home and abroad, and only several sections of patents also majority are to adopt the synthetic Carperitide of gene recombination technology, and the patent that relates to Carperitide purifying process only has one section, publication number CN102382188A.This patent has been announced a kind of Carperitide purifying process that adopts phosphoric acid buffer purifying, acetate to turn salt, but with in phosphate-buffered salt purge process, impurity is difficult to be effectively controlled, and then affects purity and yield; Turn with acetic acid in the process of salt, find Carperitide hydrochloride less stable.
The method purifying Carperitide that adopts prior art CN102382188A, purity can only reach 98.5%, total recovery approximately 32.1% left and right, the more difficult purity of patent medicine and the yield that industrialization is produced of reaching.The present invention designs new purification schemes, selects new flow phase system, adopts vitriol purifying, hydrochloric acid turns salt, and purity is reached more than 99.65%, and yield is brought up to more than 40%, the stability that has significantly improved yield, purity and Geng Gao, is easier to pharmacy use and suitability for industrialized production.
Summary of the invention
The present invention is directed to existing defective workmanship, as unstable in acetate, impurity is difficult to separate, is unfavorable for amplifying and produces etc., aims to provide that a kind of yield with low cost, high, technological process are simple, sample impurity is few and stablize controlled, to be conducive to realize industrialization Carperitide purifying process.
For achieving the above object, the present invention takes following technical scheme: a kind of purification process of Carperitide, comprises the following steps:
Step 1): by the aqueous solution sample dissolution (this step aftermentioned is referred to as " sample preparation ") containing organic solvent, prepare the solution that concentration is 100g/L;
In step 1), described sample dissolution organic solvent used is the organic solvent that polarity is strong, comprises methyl alcohol, acetonitrile, dimethyl sulfoxide (DMSO) (DMSO) or Virahol etc., wherein preferred DMSO, the volume by volume concentration of organic solvent is 3% ~ 18%, and preferably 5%-15%, is more preferably 8%-12%.
Applicant finds, due to the feature of this body structure of Carperitide composition, easily contains various not exclusively synthetic or racemization impurity in crude product, and therefore the solubilising reagent of thick peptide sample requires very harsh.After applicant's repetition test, find, organic solvent content is at 3% ~ 18%(v/v) in scope time, sample dissolution is better, more than concentration can reach 20g/L, organic solvent content is at 8%-12%(v/v) when scope, more than best results concentration can reach 40g/L; And find in the time groping different organic reagent, the organic reagent of the stronger polarity such as DMSO, methyl alcohol, acetonitrile, Virahol is better to the solute effect of sample, wherein again with DMSO best results, more than concentration can reach 50g/L.
More than sample solution concentration reaches 20g/L, be conducive to loading and the later separation of sample, preferably the solution of 100g/L.
Step 2): taking octadecylsilane chemically bonded silica as stationary phase, taking the sulfuric acid buffering salt containing perchloric acid as A phase, acetonitrile is B phase, gradient: volume by volume concentration B% is 17%～37%, and sample introduction carries out gradient elution (this step aftermentioned is referred to as " purifying ");
Step 2): use this eluent system, can effectively ensure separating effect and purification yield, wherein, particularly when B% in eluent system is not in 17%～37% scope, easily cause not hanging column or go out in advance of sample, do not reach separating effect (this step aftermentioned is referred to as " purifying ").Prior art CN102382188A purification process yield is low, purity can reach 98.5%, and purification yield of the present invention can reach more than 70%, and yield has significantly raising, purity more than 99.5% and arbitrary impurity be not more than 0.1%, can be used for staking-out work standard substance and carry out medicine registration and declare.
Step 2) in, described sulfuric acid buffering salt is for being added with 0.1%-0.8%(v/v) the 10mmol/L-50mmol/L sulfuric acid buffer salt solution of perchloric acid.Carperitide is carried out in purge process, applicant analyzes, contrasts the difference phase system that flows, and looks for best moving phase system.In process, find, what most of moving phase systems can only be simple removes partial impurities, once in purge process in order to ensure purity, can reach just often will repeatedly carry out repeatedly loaded down with trivial details recovery purifying the purity meeting the requirements; Through repeatedly the groping of applicant, finally find to add 0.1%-0.8%(v/v) the 10mmol/-50mmol/L vitriol buffer system of perchloric acid, to removing the impurity successful in thick peptide, only need purifying, once reclaim, save multistep repetitive operation.
Step 2) in, described sulfuric acid buffering salt comprises ammonium sulfate or sulfuric acid triethylamine.Sodium hydroxide or potassium hydroxide or ammoniacal liquor tune pH2.0-3.0 for sulfuric acid buffer salt solution.Applicant finds, Carperitide is for comparatively harshness of the requirement of pH value, and pH value was higher than 5 o'clock, and sample is easily separated out, and pH value is lower than 1 o'clock, sample stability variation, and at room temperature 1 hour, HPLC purity can reduce approximately 2% left and right.Through repetition test, while finding pH2.0-3.0, sample stability the best, at room temperature 1 hour, HPLC purity is almost constant.
Step 3): Carperitide vitriol is changed into hydrochloride, adopt Reversed phase HPLC method, taking octadecylsilane chemically bonded silica as stationary phase, gradient elution, freeze-drying obtains Carperitide (this step aftermentioned is referred to as " turning salt ").
In step 3), the described salt that turns comprises two steps: turn salt 20min with the aqueous solution of hydrochloride buffer salt and the mixing solutions of acetonitrile of volume ratio 95:5, in the aqueous solution of described hydrochloride buffer salt, the volume by volume concentration of hydrochloride buffer salt is that 0.02%-0.1%, pH value scope are 2.0-4.5; The aqueous hydrochloric acid that is 0.02%-0.5% with volume by volume concentration is afterwards A phase, and acetonitrile is B phase, taking volume by volume concentration B% as 5%～25%, carries out gradient elution 30min.
Applicant finds, hydrochloride buffer salt concn is lower than 0.02% time, or higher than 0.1% time, in unstable at room temperature 1 hour of sample, HPLC purity can reduce approximately 2% left and right.In hydrochloric acid system, when hydrochloride buffer salt pH value scope is 2.0-4.5, sample is all very stable, and at room temperature 1 hour, HPLC purity is almost constant.
Wherein, hydrochloric acid ammonium: the aqueous hydrochloric acid that configuration volume ratio is 0.02% ~ 0.1%, with ammoniacal liquor adjust pH 2.0 ~ 4.5.In the present invention, finding to turn salt, to turn salt lower than 20 minutes not thorough, do not reach and turn salt requirement.Exceed 20 minutes and can cause the production time to extend and material waste, increase production cost.
In step 3), the gradient of described reversed-phase HPLC, volume by volume concentration B% is 5%～25%, carries out gradient elution, the time is 30min.Applicant finds; in this eluent system, B% cannot rinse from chromatographic column lower than 5% sample, though B% can get off sample wash higher than 25%, can not improve its purity and can cause the usage quantity of organic phase to increase; increase production cost, particularly evident in the time of large-scale production.
Wash-out is lower than the separating effect that can reduce this step for 30 minutes, purity cannot reach 99.5% or more than, more than 30 minutes can make the production time extend and increase the usage quantity of material, thereby causes the increase of production cost.
This step of the present invention has not only carried out turning salt and desalination, but also purity is brought up to more than 99.5%.
In specification sheets, v/v refers to volume by volume concentration, and the two uses identical volume unit, for example mL.
Using this wash-out wash-out is according to the polarity of Carperitide itself, obtains through a large amount of experiment screenings.Can increase the purifying time if concentration is lower, increase purifying cost; If what more higher position can not be good turns salt effect.
Present method is passed through purifying, removing respectively impurity in Carperitide crude product makes purity be greater than 99.65%, is singly assortedly less than 0.1% and change into stable hydrochloride, process stabilizing is controlled, purity is high, productive rate is high, and good stability has practical value and application prospect widely.
Brief description of the drawings
Fig. 1: adopt embodiment 2,7,14 methods to obtain Carperitide essence peptide spectrogram
Fig. 2: adopt embodiment 2,7,14 methods to obtain data corresponding to the thick peptide spectrogram of Carperitide
Embodiment
Embodiment 1 sample preparation
Dissolve thick peptide according to the concentration of 100g/L with the DMSO aqueous solution of volume ratio 3%, stir that sample is dissolved is completely rear with membrane filtration, collect filtrate.
Dissolve thick peptide according to the concentration of 100g/L with the acetonitrile solution of volume ratio 10%, stir that sample is dissolved is completely rear with membrane filtration, collect filtrate.
Dissolve thick peptide according to the concentration of 100g/L with the methanol aqueous solution of volume ratio 18%, stir that sample is dissolved is completely rear with membrane filtration, collect filtrate.
Embodiment 4 sample preparation
Dissolve thick peptide according to the concentration of 100g/L with the DMSO aqueous solution of volume ratio 10%, stir that sample is dissolved is completely rear with membrane filtration, collect filtrate.
Embodiment 5 sample preparation
Dissolve thick peptide according to the concentration of 100g/L with the isopropanol water solution of volume ratio 10%, stir that sample is dissolved is completely rear with membrane filtration, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5 cm × 25 cm.Moving phase: A phase: be added with the 10mmol/L vitriol buffering (v/v) of 0.1% perchloric acid, adjust pH2.0 with sodium hydroxide; B phase: acetonitrile, flow velocity: 60-80 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 1.5g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Embodiment 7 purifying
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5 cm × 25 cm.Moving phase: A phase: be added with the 25mmol/L vitriol buffering (v/v) of 0.3% perchloric acid, adjust pH2.5 with potassium hydroxide; B phase: acetonitrile, flow velocity: 60-80 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 1.5g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Embodiment 8 purifying
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5 cm × 25 cm.Moving phase: A phase: be added with the 50mmol/L vitriol buffering (v/v) of 0.8% perchloric acid, adjust pH3.0 with ammoniacal liquor; B phase: acetonitrile, flow velocity: 60-80 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 1.5g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Embodiment 9 purifying
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10 cm × 25 cm.Moving phase: A phase: be added with the 20mmol/L vitriol buffering (v/v) of 0.3% perchloric acid, use sodium hydroxide pH3.0; B phase: acetonitrile, flow velocity: 220-250 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 15-20g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10 cm × 25 cm.Moving phase: A phase: be added with the 50mmol/L vitriol buffering (v/v) of 0.5% perchloric acid, adjust pH2.0 with potassium hydroxide; B phase: acetonitrile, flow velocity: 220-250 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 15-20g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Embodiment 11 purifying
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15 cm × 30cm.Moving phase: A phase: be added with the 45mmol/L vitriol buffering (v/v) of 0.25% perchloric acid, adjust pH2.6 with ammoniacal liquor; B phase: acetonitrile, flow velocity: 450-500 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 15-20g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Embodiment 12 purifying
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30 cm × 30cm.Moving phase: A phase: be added with the 32mmol/L vitriol buffering (v/v) of 0.23% perchloric acid, adjust pH2.0 with potassium hydroxide; B phase: acetonitrile, flow velocity: 1900-2200 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 60-855g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Embodiment 13 purifying
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30 cm × 30 cm.Moving phase: A phase: be added with the 45mmol/L vitriol buffering (v/v) of 0.18% perchloric acid, adjust pH2.5 with ammoniacal liquor; B phase: acetonitrile, flow velocity: 1900-2200 ml/min, gradient: B%:18%～28%, linear gradient elution 30min; Detect wavelength: 230 nm.Sample size is 60-85g.Collect object peak, the Carperitide solution of collection vacuum rotary steam under the condition of 30 DEG C of water temperatures is concentrated into after approximately 15 mg/mL for subsequent use.
Embodiment 14 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5 cm × 25 cm.Moving phase: A phase: 0.02% aqueous hydrochloric acid (v/v); B phase: acetonitrile; C phase: 0.02% salt acid amide, pH2.0, flow velocity: 80ml/min, detects wavelength: 230 nm.Sample size is 1.5g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.75%, other impurity is all less than 0.07%, and yield is 42.5%.
Embodiment 15 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5 cm × 25 cm.Moving phase: A phase: 0.05% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.05% salt acid amide, pH2.5, flow velocity: 60 ml/min, detect wavelength: 230 nm.Sample size is 1.5g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.68%, other impurity is all less than 0.08%, and yield is 41.3%.
Embodiment 16 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5 cm × 25 cm.Moving phase: A phase: 0.1% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.08% salt acid amide, pH3.0, flow velocity: 80 ml/min; Detect wavelength: 230 nm.Sample size is 1.5g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.68%, other impurity is all less than 0.08%, and yield is 41.5%.
Embodiment 17 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10 cm × 25 cm.Moving phase: A phase: 0.06% aqueous hydrochloric acid (v/v); B phase: acetonitrile, flow velocity: 220ml/min, detects wavelength: 230 nm.Sample size is 10-15g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.66%, other impurity is all less than 0.08%, and yield is 40.8%.
Embodiment 18 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10 cm × 25 cm.Moving phase: A phase: 0.03% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.10% salt acid amide, pH4.5, flow velocity: 250 ml/min, detect wavelength: 230 nm.Sample size is 15-20g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.72%, other impurity is all less than 0.08%, and yield is 39.5%.
Embodiment 19 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15 cm × 30 cm.Moving phase: A phase: 0.09% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.07% salt acid amide, pH3.2, flow velocity: 400ml/min, detects wavelength: 230 nm.Sample size is 15-20g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99. 70%, other impurity is all less than 0.08%, and yield is 40.5%.
Embodiment 20 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15 cm × 30 cm.Moving phase: A phase: 0.65% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.06% salt acid amide, pH2.0, flow velocity: 450 ml/min, detect wavelength: 230 nm.Sample size is 15-20g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.70%, other impurity is all less than 0.08%, and yield is 39.7%.
Embodiment 21 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30 cm × 30 cm.Moving phase: A phase: 0.32% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.05% salt acid amide, pH2.5, flow velocity: 1900ml/min, detects wavelength: 230 nm.Sample size is 60-85g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.72%, other impurity is all less than 0.08%, and yield is 40.6%.
Embodiment 22 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm × 30 cm.Moving phase: A phase: 0.10% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.02% salt acid amide, pH2.0, flow velocity: 2200 ml/min, detect wavelength: 230 nm.Sample size is 60-85g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.70%, other impurity is all less than 0.08%, and yield is 40.2%.
Embodiment 23 turns salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30 cm × 30 cm.Moving phase: A phase: 0.08% aqueous hydrochloric acid (v/v); B phase: acetonitrile, C phase: 0.68% salt acid amide, pH2.3, flow velocity: 2000 ml/min, detect wavelength: 230 nm.Sample size is 60-85g.
Step: with 5%B+95%C(V/V) turn after salt 20min, with 5%B+95%A～25% B+75%A(V/V), linear gradient elution 30min; Collect object peak, after vacuum rotary steam is concentrated into about 50-200 mg/mL under 30 DEG C of conditions of water temperature by the Carperitide solution of collection, go to 50ml cillin bottle.After lyophilize, can obtain the Carperitide that purity is greater than 99.70%, other impurity is all less than 0.08%, and yield is 39.8%.
Claims (4)
1. a purification process for Carperitide, comprises the following steps:
Step 1): the aqueous solution sample dissolution of the organic solvent that the polarity that is 3%～18% with volume by volume concentration is strong, prepare the solution that concentration is 100g/L, described organic solvent is methyl alcohol, acetonitrile, dimethyl sulfoxide (DMSO) or Virahol;
Step 2): taking octadecylsilane chemically bonded silica as stationary phase, taking the 10mmol/L-50mmol/L sulfuric acid buffer salt solution of perchloric acid that adds volume ratio 0.1%-0.8% as A phase, acetonitrile is B phase, volume by volume concentration B% is 18%～28%, the solution that sample introduction step 1) makes, carries out gradient elution, described step 2) in, sulfuric acid buffering salt refers to ammonium sulfate or sulfuric acid triethylamine, sodium hydroxide or potassium hydroxide or ammoniacal liquor tune pH2.0-3.0 for sulfuric acid buffer salt solution;
Step 3): Carperitide vitriol is changed into hydrochloride, adopt Reversed phase HPLC method, taking octadecylsilane chemically bonded silica as stationary phase, gradient elution, freeze-drying obtains Carperitide, described step 3) comprises two steps: turn salt 20min with the aqueous solution of hydrochloride buffer salt and the mixing solutions of acetonitrile of volume ratio 95:5, in the aqueous solution of described hydrochloride buffer salt, the volume by volume concentration of hydrochloride buffer salt is that 0.02%-0.1%, pH value scope are 2.0-4.5; The aqueous hydrochloric acid that is 0.02%-0.5% with volume by volume concentration is afterwards A phase, and acetonitrile is B phase, taking volume by volume concentration B% as 5%～25%, carries out gradient elution 30min.
2. method according to claim 1, is characterized in that: in described step 1), organic solvent volume ratio content is 5%-15%.
3. method according to claim 1, is characterized in that: in described step 1), organic solvent volume ratio content is 8%-12%.
4. method according to claim 1, is characterized in that: described hydrochloride buffer salt is salt acid amide.
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| WO2009098707A1 (en) * | 2008-02-06 | 2009-08-13 | Biocon Limited | A method of purifying a peptide |
| CN101463080B (en) * | 2009-01-09 | 2011-09-14 | 深圳翰宇药业股份有限公司 | Method for purifying nesiritide |
| CN102250235A (en) * | 2011-06-23 | 2011-11-23 | 成都圣诺科技发展有限公司 | Preparation method of nesiritide |
| CN102382188B (en) * | 2011-11-07 | 2014-01-22 | 深圳翰宇药业股份有限公司 | Method for preparing carperitide acetate |
| CN102584953B (en) * | 2012-02-09 | 2014-01-01 | 深圳翰宇药业股份有限公司 | Purification method for atosiban |
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