CN103848893B - A kind of purification process of Linaclotide - Google Patents
A kind of purification process of Linaclotide Download PDFInfo
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Abstract
The invention belongs to field of pharmaceutical chemistry technology, disclose the purification process of a kind of Linaclotide.Purification process of the present invention is with porous graphite carbon for fixing phase, three-dimensional selective agent is made in conjunction with flowing mutually adds certain density γ cyclodextrin, high performance liquid chromatography peptide thick to Linaclotide is used to carry out isolated and purified, then utilizing high performance liquid chromatography to remove and change into acetate by the γ cyclodextrin in Linaclotide eluent prepared for purification and phosphate, lyophilizing prepares Linaclotide fine peptide.Purification process of the present invention is simple to operate, so that running cost is low.In addition, purification process Linaclotide yield of the present invention is high, thus it is suitable for the large-scale industrialized production of Linaclotide, and the purified Linaclotide fine peptide purity obtained is up to more than 99%, purity is high, impurity content is low, therefore has considerable economical and practical value and is widely applied prospect.
Description
Technical field
The invention belongs to field of pharmaceutical chemistry technology, particularly relate to the purification process of a kind of Linaclotide.
Background technology
Constipation is a kind of commonly encountered diseases, can be divided into organic and functional two classes from the cause of disease.Wherein functional constipation be by
Intestinal Mucosal Injury in Patients Undergoing is wriggled caused by abnormal and non-bowel genetic morphology pathological changes.Functional constipation some patients is chronic slow-transit constipation
With irritable bowel syndrome (IBS) type constipation.Chronic slow-transit constipation, is due to intestinal contraction hypomotility, make feces from caecum to
The movement of rectum is slowed down, or causes due to the asynergic movement of left hemicolon.It is most commonly in young woman, after preadolescence
Occur, it is characterized by that defecation frequency reduces (defecation is less than 1 time weekly), few awareness of defecation, excrement matter is hard, thus difficult defecation；Anus is straight
Without feces or touch hard feces when intestinal refers to inspection, and the contracting anus of external anal sphincter and firmly bowel movement function are normal；Full gastrointestinal or
Colonic adaption extends；Lack the evidence of Treatment of Outlet Obstruction Type, discharge test such as air bag and technique of anorectal manometry is normal.IBS belongs to
Functional gastrointestinal disorder disease.Morbidity is how relevant with gastrointestinal tract dynamia exception or visceral sense hypersensitivity, the normal table of its symptom
Now for stomachache, diarrhoea or constipation and abdominal distention, have to go to the toilet.
Linaclotide (Linaclotide), is that the one that FDA (FDA) ratifies has novel work
With mechanism be used for treat chronic slow-transit constipation and the oral drugs of irritable bowel syndrome (IBS) type constipation.It is a kind of guanosine
Cyclase of acid-C agonist, can activate the GC-C receptor of enterocyte top end surface, causes intracellular and extracellular loop guanosine
Acid increases.Net effect is chlorine and Bicarbonate secretion entrance enteric cavity increase, and then causes liquid secretion to increase and will pass through greatly
Accelerate.Linaclotide (150 ~ 300 μ g/d) can make spontaneous defecation quantity weekly increase, and improves stool, alleviates stool
Firmly degree, and the order of severity of constipation.Therefore Linaclotide can be as one of line medication for treatment functional constipation, city
Field has a extensive future.
Linaclotide, the polypeptide being made up of 14 aminoacid, molecular formula is C59H79N15O21S6, molecular weight is 1526.74.
Linaclotide peptide chain is included in looped three pairs of disulfide bond (three pairs of disulfide bond are 1-6,2-10,5-13) between Cys and Cys,
Its structural formula is as follows:
At present, Linaclotide is mainly obtained by solid phase synthesis technique.Owing to Linaclotide has the existence of three pairs of disulfide bond,
No matter it is to use solid phase coupling solid phase location oxidation technology route, or the technology path of liquid phase oxidation after employing solid phase coupling
Synthesize, all can produce the impurity of other disulfide bond pairing.But only Linaclotide three correctly matching just to disulfide bond
Curative effect can be played, therefore remove these incorrect pairing impurity by purification and other impurity becomes inevitable choice.Due to profit that
In the peptide crude product of Lip river, disulfide bond incorrect pairing amount of impurities is numerous and its chemical property is the most closely similar with Linaclotide, therefore profit
The isolated and purified technological difficulties become in this medicine preparation technology of that Lip river peptide, the Linaclotide of preparation is pure especially on a large scale
Change and become one of bottleneck restricting its industrialization.So far there are no can the report of purification process of scale separation Linaclotide
Road.
Summary of the invention
In view of this, it is an object of the invention to provide the purification process of a kind of Linaclotide.
For realizing the purpose of the present invention, the present invention adopts the following technical scheme that
The purification process of a kind of Linaclotide, comprises the following steps:
Step 1: using high performance liquid chromatography peptide thick to Linaclotide to be purified, described high performance liquid chromatography is with many
Hole graphitized carbon is fixing phase, after thick peptide solution loading, with the phosphate buffered solution containing gamma-cyclodextrin for mobile phase A phase,
Carry out gradient elution with acetonitrile for Mobile phase B mutually, collect eluent；
Step 2: use high performance liquid chromatography that step 1 gained eluent is turned salt, described high performance liquid chromatography
With octadecylsilane chemically bonded silica for fixing phase, after step 1 gained eluent loading, with glacial acetic acid aqueous solution as mobile phase A
Phase, carries out gradient elution mutually with acetonitrile for Mobile phase B, collects eluent, lyophilizing and get final product.
The thick peptide of Linaclotide of the present invention refers to use solid phase coupling solid phase location oxidation technology route or solid phase even
After connection the technology path of liquid phase oxidation and other method prepare, can not without Linaclotide or the purity of refinement treatment
Meet medicinal Linaclotide.
Owing to Linaclotide has the existence of three pairs of disulfide bond to have more stable solid space structure, this matching method makes
Linaclotide has different solid space structures and surface hydrophobic from other incorrect pairing impurity.The present invention utilizes porous
Graphitized carbon is fixing to be had anti-phase hydrophobic interaction mutually and is similar to the macromole of polycyclic aromatic hydrocarbon and geometric separation because carbon atom is formed
The advantageous feature of isomer, makees three-dimensional selective agent in conjunction with adding certain density gamma-cyclodextrin in flowing mutually, uses efficiently
Liquid chromatography carries out the isolated and purified of Linaclotide, with porous graphite carbon for fixing phase, after thick peptide solution loading, to contain
The phosphate buffered solution of gamma-cyclodextrin is mobile phase A phase, carries out gradient elution mutually with acetonitrile for Mobile phase B, collects eluting
Liquid.
Those skilled in the art can select different size size (pillar diameter according to the amount of the thick peptide of purification Linaclotide
× length) chromatography such as 5cm × 25cm specification chromatographic column, 10cm × 25cm specification chromatographic column or 30cm × 25cm specification
Chromatographic column.
Preferably, based on g/mL, mobile phase A described in step 1 mutually in gamma-cyclodextrin concentration be 0.2% ~ 1.0%.More preferably
Being 0.3% ~ 0.6%, in certain embodiments, described gamma-cyclodextrin concentration is 0.2%, and i.e. every 20g gamma-cyclodextrin adds 10L phosphorus
Hydrochlorate buffer solution.In certain embodiments, described gamma-cyclodextrin concentration is 0.3%；In certain embodiments, described γ-ring
Dextrin concentration is 0.3%；In certain embodiments, described gamma-cyclodextrin concentration is 0.4%；In certain embodiments, described γ-
Cyclodextrin concentration is 0.5%；In certain embodiments, described gamma-cyclodextrin concentration is 0.6%；In certain embodiments, described
Gamma-cyclodextrin concentration is 1.0%.
Preferably, mobile phase A described in step 1 mutually in phosphatic molar concentration be 10mM ~ 30mM.More preferably 20mM
~25mM。
Preferably, mobile phase A described in step 1 mutually in phosphate selected from sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate,
One or more in potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium phosphate or ammonium dihydrogen phosphate.
Preferably, described in step 1, the pH of mobile phase A phase solution is 2.0 ~ 4.0.More preferably 2.5 ~ 3.0.
Preferably, gradient described in step 1 according to the percentage ratio of Mobile phase B phase is Those skilled in the art suitably can adjust according to staying the time.Ladder described in step 1 in certain embodiments
Degree isGradient described in step 1 is in certain embodimentsGradient described in step 1 is in certain embodimentsGradient described in step 1 is in certain embodimentsGradient described in step 1 is in further embodiments
Purification process step 2 of the present invention utilizes high performance liquid chromatography to be stuck with paste by the γ in step 1 gained eluent-ring
Essence and phosphate remove and change into acetate.With octadecylsilane chemically bonded silica for fixing phase, step 1 gained eluent loading
After, with glacial acetic acid aqueous solution for mobile phase A phase, carrying out gradient elution mutually with acetonitrile for Mobile phase B, collect eluent, lyophilizing obtains
To Linaclotide.
Equally, those skilled in the art can according to the amount of purification eluent, select different size size (pillar diameter ×
Length) chromatography such as 5cm × 25cm specification chromatographic column, 10cm × 25cm specification chromatographic column or 30cm × 25cm specification color
Spectrum post.Those skilled in the art can be different according to the diameter of purification chromatographic column, select different applied sample amounts.
Purification process step 2 glacial acetic acid aqueous solution of the present invention is mobile phase A phase, carries out mutually with acetonitrile for Mobile phase B
Gradient elution.
As preferably, mobile phase A described in step 2 mutually in glacial acetic acid aqueous solution in the concentration expressed in percentage by volume of glacial acetic acid be
0.1%~0.5%.More preferably 0.2% ~ 0.4%.It is 0.25% in certain embodiments；It is 0.3% in certain embodiments.
Preferably, gradient described in step 2 is
Preferably, also include before lyophilizing described in purification process step 2 of the present invention that the eluent to collecting concentrates
Step.Described concentration is preferably and is concentrated by prepared Linaclotide vacuum rotary steam.
The water that purification process of the present invention is used is pure water, and meets water for injection standard, the most ultrapure
Water；Glacial acetic acid used in the present invention is analytically pure glacial acetic acid；Acetonitrile used in the present invention is analytically pure acetonitrile, preferably
For chromatographically pure.
Purification process of the present invention is with porous graphite carbon for fixing phase, certain density in conjunction with adding in flowing mutually
Gamma-cyclodextrin makees three-dimensional selective agent, uses high performance liquid chromatography peptide thick to Linaclotide to carry out isolated and purified, then utilizes height
Gamma-cyclodextrin and phosphate in the Linaclotide eluent that purification is prepared by effect liquid phase chromatogram remove and change into acetate, freeze
Drying Linaclotide fine peptide.Purification process of the present invention is simple to operate, so that running cost is low.Additionally, the present invention
Described purification process Linaclotide yield is high, thus is suitable for the large-scale industrialized production of Linaclotide, and purified
The Linaclotide fine peptide purity arrived is up to more than 99%, and purity is high, impurity content is low, therefore has considerable economical and practical value
Be widely applied prospect.
Accompanying drawing explanation
Fig. 1 shows the high performance liquid chromatography testing result figure of the prepared Linaclotide fine peptide of embodiment 1.
Detailed description of the invention
The embodiment of the invention discloses the purification process of a kind of Linaclotide.In those skilled in the art can use for reference herein
Hold, be suitably modified technological parameter and realize.Special needs to be pointed out is, all similar replacements and change are to those skilled in the art
For be apparent from, they are considered as being included in the present invention.The method of the present invention is carried out by preferred embodiment
Describing, method described herein substantially can be modified in without departing from present invention, spirit and scope by related personnel
Or suitably change and combination, realize and apply the technology of the present invention.
In order to be further appreciated by the present invention, below in conjunction with embodiment, the present invention is described in detail.
In the examples below, the mensuration of Linaclotide content and purity is according to high performance liquid chromatography (Chinese Pharmacopoeia
Two annex V D of version in 2010) carry out.Concrete operations are as follows:
Chromatographic condition and system suitability: chromatographic column used makees filler, chromatographic column specification with porous graphite carbon
Being 4.6 × 150mm, the particle diameter of the porous graphite carbon filled is 3 μm；(di(2-ethylhexyl)phosphate is taken with 30mmol/L sodium phosphate buffer
Hydrogen sodium 4.0g, adds water to 1000mL, is 2.5 with phosphorus acid for adjusting pH) it is mobile phase A phase, with trifluoroacetic acid aqueous solution for Mobile phase B phase,
According to the form below 1 carries out gradient elution；Flow velocity is 1.0mL/ minute, and detection wavelength is 215nm, and column temperature is 60 DEG C；Number of theoretical plate is by profit
That Luo Taifeng calculates should be not less than 5000.
Gradient in the chromatography of table 1 Linaclotide assay
| Time (minute) | Mobile phase A phase (%) | Mobile phase B phase (%) |
| 0-20 | 90-75 | 10-25 |
| 20-25 | 75-50 | 20-50 |
| 25-26 | 50-90 | 50-10 |
| 26-30 | 90 | 10 |
The assay method of Linaclotide content: take Linaclotide crude product appropriate, add water and make containing 2.0mg/mL Linaclotide
The solution of crude product, as need testing solution, the most accurately weighs appropriate Linaclotide reference substance, add water make containing 1.0mg/mL profit that
The solution of Lip river peptide reference substance is as reference substance solution；Take reference substance solution and each 10 μ L of need testing solution are injected separately into liquid chromatograph
Instrument, records chromatogram, calculates by external standard method with peak area, i.e. obtains the content of Linaclotide.
The assay method of Linaclotide purity: take Linaclotide appropriate, add water and make the solution containing about 1.0mg/mL；Take each
10 μ L solution inject chromatograph of liquid, record chromatogram, with area normalization method computer chromatography purity.
Embodiment 1:
By thick for Linaclotide peptide 4.0g(1.81g Han Linaclotide) dissolve by purified water 200mL, filter, collect filtrate standby
With.
Purification chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in porous
Graphitized carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.2%(g/mL, W/V) the 30mM biphosphate sodium water solution of gamma-cyclodextrin, adjust the pH to be with phosphoric acid
2.0.Process for preparation: weigh 20g gamma-cyclodextrin and 36g sodium dihydrogen phosphate, appropriate purified water crosses 0.45 μm filter membrane mistake after dissolving
Filter, filtrate all collects 10L serum bottle, adds purified water to 10L scale, adjusts pH to be 2.0 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Applied sample amount: 100mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction at twice, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in anti-phase
C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.10% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 200mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 20mL.
White powdery solids fine peptide 1.46g is obtained after lyophilizing.The result of the fine peptide that high performance liquid chromatography detection purification prepares
Seeing Fig. 1, wherein abscissa represents the operation time in minutes, and vertical coordinate represents peak height.Statistical result is shown in Table 2.
The chromatograph testing result of table 2 Linaclotide solid fine peptide
| Peak is numbered | Retention time (minute) | Area | Peak height | Area percentage (%) |
| 1 | 12.267 | 10855 | 704 | 0.02 |
| 2 | 13.881 | 30061 | 1637 | 0.05 |
| 3 | 14.300 | 26588 | 1552 | 0.04 |
| 4 | 14.919 | 20311 | 1057 | 0.03 |
| 5 | 15.417 | 16736 | 908 | 0.03 |
| 6 | 17.466 | 40801 | 1749 | 0.06 |
| 7 | 18.712 | 34535 | 2435 | 0.05 |
| 8 | 19.539 | 66247270 | 2339391 | 99.45 |
| 9 | 21.228 | 31578 | 1445 | 0.05 |
| 10 | 21.871 | 93224 | 3056 | 0.14 |
| 11 | 24.880 | 58395 | 1968 | 0.09 |
From table 2 result, the Linaclotide fine peptide purity that purification prepares is 99.45%, and each impurity content is respectively less than
0.15%.Purification yield is that 80.7%(is with Linaclotide cubage in crude product), total recovery is 36.5%.
Embodiment 2:
By thick for Linaclotide peptide 6.0g(2.71g Han Linaclotide) dissolve by purified water 300mL, filter, collect filtrate standby
With.
Purification chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in porous
Graphitized carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.5%(W/V) the 20mM biphosphate sodium water solution of gamma-cyclodextrin, adjust pH to be 2.5 with phosphoric acid.Join
Process processed: weigh 50g gamma-cyclodextrin and 24g sodium dihydrogen phosphate, appropriate purified water crosses 0.45 μm membrane filtration, filtrate after dissolving
All collect 10L serum bottle, add purified water to 10L scale, adjust pH to be 2.5 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 100mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction in three times, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in anti-phase
C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.10% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 200mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 20mL.
White powdery solids fine peptide 2.13g is obtained after lyophilizing.Purity 99.47%, each impurity content is respectively less than 0.15%.Purification
Yield 78.6%(is with Linaclotide cubage in crude product), total recovery 35.5%.
Embodiment 3:
By thick for Linaclotide peptide 6.0g(2.71g Han Linaclotide) dissolve by purified water 300mL, filter, collect filtrate standby
With.
Purification chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in porous
Graphitized carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 1.0%(W/V) the 20mM ammonium dihydrogen phosphate aqueous solution of gamma-cyclodextrin, adjust pH to be 3.0 with phosphoric acid.Join
Process processed: weigh 100g gamma-cyclodextrin and 23g ammonium dihydrogen phosphate, appropriate purified water crosses 0.45 μm membrane filtration, filtrate after dissolving
All collect 10L serum bottle, add purified water to 10L scale, adjust pH to be 3.0 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 2.0g (100mL).
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction in three times, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in anti-phase
C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.30% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 200mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 20mL.
White powdery solids fine peptide 2.17g is obtained after lyophilizing.Purity 99.36%, each impurity content is respectively less than 0.15%.Purification
Yield 80.1%(is with Linaclotide cubage in crude product), total recovery 36.1%.
Embodiment 4:
Linaclotide is aoxidized concentrated solution crude product 200mL(2.82g Han Linaclotide), filter, collect filtrate standby.
Purification chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in porous
Graphitized carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.4%(W/V) the 30mM biphosphate sodium water solution of gamma-cyclodextrin, adjust pH to be 3.0 with phosphoric acid.Join
Process processed: weigh 40g gamma-cyclodextrin and 36g sodium dihydrogen phosphate, appropriate purified water crosses 0.45 μm membrane filtration, filtrate after dissolving
All collect 10L serum bottle, add purified water to 10L scale, adjust pH to be 3.0 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 100mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction at twice, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in anti-phase
C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.50% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 200mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 20mL.
White powdery solids fine peptide 2.25g is obtained after lyophilizing.Purity 99.37%, each impurity content is respectively less than 0.15%.Purification
Yield 79.7%(calculates in order to that Lip river peptide content).
Embodiment 5:
Linaclotide is aoxidized concentrated solution crude product 400mL(5.64g Han Linaclotide), filter, collect filtrate standby.
Purification chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in porous
Graphitized carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.6%(W/V) the 25mM sodium phosphate aqueous solution of gamma-cyclodextrin, adjust pH to be 4.0 with phosphoric acid.Prepared
Journey: weighing 60g gamma-cyclodextrin and 41g sodium phosphate, cross 0.45 μm membrane filtration after the dissolving of appropriate purified water, filtrate is all collected
To 10L serum bottle, add purified water to 10L scale, adjust pH to be 4.0 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 100mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction in four times, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in anti-phase
C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.25% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 200mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 40mL.
White powdery solids fine peptide 4.55g is obtained after lyophilizing.Purity 99.24%, each impurity content is respectively less than 0.15%.Purification
Yield 80.6%(calculates in order to that Lip river peptide content).
Embodiment 6:
Linaclotide is aoxidized concentrated solution crude product 400mL(5.64g Han Linaclotide), filter, collect filtrate standby.
Purification chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in porous
Graphitized carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.3%(W/V) the 20mM ammonium dihydrogen phosphate aqueous solution of gamma-cyclodextrin, adjust pH to be 2.5 with phosphoric acid.Join
Process processed: weigh 30g gamma-cyclodextrin and 23g ammonium dihydrogen phosphate, appropriate purified water crosses 0.45 μm membrane filtration, filtrate after dissolving
All collect 10L serum bottle, add purified water to 10L scale, adjust pH to be 2.5 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 100mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction in four times, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Waters 2545；Chromatographic column: 50 × 250mm, built-in anti-phase
C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 80mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.40% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 200mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 40mL.
White powdery solids fine peptide 4.42g is obtained after lyophilizing.Purity 99.43%, each impurity content is respectively less than 0.15%.Purification
Yield 78.4%(calculates in order to that Lip river peptide content).
Embodiment 7:
Linaclotide is aoxidized concentrated solution crude product 2000mL(56.4g Han Linaclotide), filter, collect filtrate standby.
Purification chromatographic condition: high performance liquid chromatograph model: Novasep LC150；Chromatographic column: 100 × 250mm, built-in
Porous graphite carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 250mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.6%(W/V) the 20mM biphosphate sodium water solution of gamma-cyclodextrin, adjust pH to be 2.5 with phosphoric acid.Join
Process processed: weigh 120g gamma-cyclodextrin and 48g sodium dihydrogen phosphate, appropriate purified water crosses 0.45 μm membrane filtration, filter after dissolving
Liquid all collects 20L serum bottle, adds purified water to 20L scale, adjusts pH to be 2.5 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 500mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.
Sample introduction in four times, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Novasep LC150；Chromatographic column: 100 × 250mm, built-in
Anti-phase C18 chromatograph packing material, the particle diameter of this filler is 10 μm.；
Flow velocity: 250mL/ minute.；
Detection wavelength: 280nm.
Mobile phase A phase: 0.20% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 1000mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 200mL.
White powdery solids fine peptide 45.2g is obtained after lyophilizing.Purity 99.37%, each impurity content is respectively less than 0.15%.Purification
Yield 80.3%(calculates in order to that Lip river peptide content).
Embodiment 8:
Linaclotide is aoxidized concentrated solution crude product 5000mL(141g Han Linaclotide), filter, collect filtrate standby.
Purification chromatographic condition: high performance liquid chromatograph model: Novasep LC150；Chromatographic column: 300 × 250mm, built-in
Porous graphite carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 2000mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.3%(W/V) the 20mM potassium dihydrogen phosphate aqueous solution of gamma-cyclodextrin, adjust pH to be 2.5 with phosphoric acid.Join
Process processed: weigh 150g gamma-cyclodextrin and 136g sodium dihydrogen phosphate, appropriate purified water crosses 0.45 μm membrane filtration, filter after dissolving
Liquid all collects 50L serum bottle, adds purified water to 50L scale, adjusts pH to be 2.5 with phosphoric acid.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 2500mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction at twice, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Novasep LC300；Chromatographic column: 300 × 250mm, built-in
Anti-phase C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 2000mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.2% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 5000mL.
Purge process: loading after chromatographic column being balanced 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 450mL.
White powdery solids fine peptide 114.6g is obtained after lyophilizing.Purity 99.37%, each impurity content is respectively less than 0.15%.Pure
Change yield 81.3%(to calculate in order to that Lip river peptide content).
Embodiment 9:
Linaclotide is aoxidized concentrated solution crude product 25L(675g Han Linaclotide), filter, collect filtrate standby.
Purification chromatographic condition: high performance liquid chromatograph model: Novasep LC150；Chromatographic column: 300 × 250mm, built-in
Porous graphite carbon is fixed phase stuffing, and the particle diameter of this filler is 7 μm；Flow velocity: 2000mL/ minute；Detection wavelength: 280nm.
Mobile phase A phase: 0.2%(W/V) the 20mM biphosphate sodium water solution of gamma-cyclodextrin, adjust pH to be 2.5 with phosphoric acid.Join
Process processed: weigh 200g gamma-cyclodextrin and 240g sodium dihydrogen phosphate, appropriate purified water crosses 0.45 μm membrane filtration, filter after dissolving
Liquid all collects 100L scale fluid reservoir, adds purified water to 100L scale, adjusts pH to be 2.5 with phosphoric acid.It is finished and repeats preparation.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Applied sample amount: 2500mL.
Purge process: loading after making chromatographic column balance 5 minutes, runs gradient-purified, and monitoring Fractional Collections purpose peak are washed
De-liquid.Collected eluent is carried out purity detecting, after the purity eluent more than or equal to 99.0% is removed major part acetonitrile
For turning salt；Reclaim after purity is less than more than or equal to 80% the eluent removing major part acetonitrile of 99.0% and repeat this purification mistake
Journey, again collects after the purity eluent more than or equal to 99.0% removes major part acetonitrile and is also used for turning salt；Purity is less than 80%
Liquid waste processing pressed by eluent.Sample introduction in ten times, repeats above operation.
Turn salt chromatographic condition: high performance liquid chromatograph model: Novasep LC300；Chromatographic column: 300 × 250mm, built-in
Anti-phase C18 chromatograph packing material, the particle diameter of this filler is 10 μm；Flow velocity: 2000mL/ minute；
Detection wavelength: 280nm.
Mobile phase A phase: 0.2% glacial acetic acid (v/v) solution.
Mobile phase B phase: trifluoroacetic acid aqueous solution.
Gradient:
Loading volume: 5000mL.
Purge process: loading after chromatographic column being balanced 5 minutes, runs gradient-purified, monitors and collect purpose peak eluent.
Purpose peak eluent vacuum rotary steam is concentrated into lyophilizing after 2.5L.
White powdery solids fine peptide 528g is obtained after lyophilizing.Purity 99.39%, each impurity content is respectively less than 0.15%.Purification
Yield 78.3%(calculates in order to that Lip river peptide content).
The explanation of above example is only intended to help to understand method and the core concept thereof of the present invention.It is right to it should be pointed out that,
For those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out
Some improvement and modification, these improve and modify in the protection domain also falling into the claims in the present invention.
Claims (9)
1. the purification process of a Linaclotide, it is characterised in that comprise the following steps:
Step 1: using high performance liquid chromatography peptide thick to Linaclotide to be purified, described high performance liquid chromatography is with porous stone
Inkization carbon is fixing phase, after thick peptide solution loading, with the phosphate buffered solution containing gamma-cyclodextrin for mobile phase A phase, with second
Nitrile is that Mobile phase B carries out gradient elution mutually, collects eluent；
Step 2: using high performance liquid chromatography that step 1 gained eluent is turned salt, described high performance liquid chromatography is with ten
Eight alkyl silane bonded silica gels are fixing phase, after step 1 gained eluent loading, with glacial acetic acid aqueous solution for mobile phase A phase, with
Acetonitrile is that Mobile phase B carries out gradient elution mutually, collects eluent, lyophilizing and get final product.
Purification process the most according to claim 1, it is characterised in that based on g/mL, mobile phase A described in step 1 mutually in γ-
Cyclodextrin concentration is 0.2%-1.0%.
Purification process the most according to claim 1, it is characterised in that mobile phase A described in step 1 mutually in phosphatic mole
Concentration is 10mM～30mM.
Purification process the most according to claim 1, it is characterised in that mobile phase A described in step 1 mutually in phosphate selected from phosphorus
In acid sodium, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium phosphate or ammonium dihydrogen phosphate
One or more.
Purification process the most according to claim 1, it is characterised in that the pH of mobile phase A phase solution described in step 1 be 2.0～
4.0。
Purification process the most according to claim 1, it is characterised in that described in step 1, gradient is in 35min, Mobile phase B phase
Percentage ratio is from 10%～20% to 30%～40%.
Purification process the most according to claim 1, it is characterised in that mobile phase A described in step 2 mutually in glacial acetic acid aqueous solution
The concentration expressed in percentage by volume of middle glacial acetic acid is 0.1%～0.5%.
Purification process the most according to claim 1, it is characterised in that gradient described in step 2 is in 20min, and flowing matches
Ratio is from mobile phase A: Mobile phase B=95:5 is to mobile phase A: Mobile phase B=95:5；Afterwards in 2min, proportion of mobile phase is from stream
Dynamic phase A: Mobile phase B=95:5 is to mobile phase A: Mobile phase B=70:30；Afterwards in 10min, proportion of mobile phase is from flowing phase
A: Mobile phase B=70:30 is to mobile phase A: Mobile phase B=70:30.
Purification process the most according to claim 1, it is characterised in that also include before lyophilizing described in step 2 eluting collected
Liquid carries out concentration step.
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| CN113683663A (en) * | 2021-09-16 | 2021-11-23 | 杭州信海医药科技有限公司 | Purification method of organism-protected polypeptide crude product |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101372504A (en) * | 2007-08-22 | 2009-02-25 | 深圳市翰宇药业有限公司 | Method for purifying desmopressin |
| CN102482326A (en) * | 2009-04-10 | 2012-05-30 | 科登制药科罗拉多公司 | Process for isolating therapeutic peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101372504A (en) * | 2007-08-22 | 2009-02-25 | 深圳市翰宇药业有限公司 | Method for purifying desmopressin |
| CN102482326A (en) * | 2009-04-10 | 2012-05-30 | 科登制药科罗拉多公司 | Process for isolating therapeutic peptide |
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| Title |
|---|
| Igor Clarot et al..Elution Characteristics of Natural Cyclodextrins on Porous Graphitic Carbon.《Journal of Chromatographic Science》.2000,第38卷(第1期),第38-45页. * |
| Jian-Qiang Fan et al..High-Performance Liquid Chromotography of Glycopeptides and Oligosacchrides on Graphitized Carbon Columns.《ANALYTICAL BIOCHEMISTRY》.1994,第219卷(第2期),第224-229页. * |
| Separation of polar compounds using carbon columns;Toshihiko Hanai;《Journal of Chromatography A》;20030314;第989卷(第2期);第183-196页 * |
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