CN110078797B - Method for refining oxytocin [4-Glu ] impurity - Google Patents
Method for refining oxytocin [4-Glu ] impurity Download PDFInfo
- Publication number
- CN110078797B CN110078797B CN201910376583.0A CN201910376583A CN110078797B CN 110078797 B CN110078797 B CN 110078797B CN 201910376583 A CN201910376583 A CN 201910376583A CN 110078797 B CN110078797 B CN 110078797B
- Authority
- CN
- China
- Prior art keywords
- oxytocin
- glu
- impurity
- phase
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a refining method of oxytocin [4-Glu ] impurities, which comprises the following steps: adopting a high performance liquid phase reverse phase chromatography to carry out reverse phase enrichment, reverse phase salt conversion and reverse phase purification on the oxytocin [4-Glu ] impurity crude product solution in sequence; the filler of the high performance liquid reverse phase chromatography is super water-resistant filler; the reversed-phase enrichment, the reversed-phase salt conversion and the reversed-phase purification are all completed in the one-step reversed-phase elution process. The method for refining oxytocin [4-Glu ] impurities has the advantages that most of waste liquid generated in the purification process is waste water, can be recycled through simple treatment of a sewage station, and is economical and environment-friendly.
Description
Technical Field
The invention relates to a refining method of oxytocin [4-Glu ] impurities.
Background
Oxytocin, also known as Oxytocin, is known as oxyytocin and has the structural formula:
the molecular formula is: c43H66N12O12S2Molecular weight of 1007.2
The oxytocin is used for induced labor, postpartum and postpartum metrorrhagia caused by uterine weakness or poor abdomen contraction; understanding placental reserve function (oxytocin rage test); it can promote milk excretion by dripping into nose. Oxytocin can indirectly stimulate uterine smooth muscle to shrink, simulate uterine contraction effect of normal delivery, and cause cervix dilatation, and uterine response to oxytocin gradually increases in the pregnancy process, and reaches peak at term. Oxytocin may also stimulate contraction of the smooth muscle of the breast, facilitating the drainage of milk from the breast, but does not increase the milk production of the breast.
In the case of a drug, the small amount of impurities contained therein is the most important cause for the side effects of the drug, so that the purity inspection is one of the important bases for ensuring the safety and effectiveness of the drug, and the content of the purity inspection is somewhat different according to the properties and characteristics of each drug, but basically involves respective inspection research on "related substances". Although the purification process of the synthesized polypeptide has been greatly improved at present, the process impurities are still important sources of the synthesized polypeptide-related substances, mainly because some process impurities (such as deletion peptides, broken peptides, oxidized peptides, products of disulfide bond exchange and the like) of the synthesized polypeptide may be very similar to the properties of the drug per se, thereby causing certain difficulty in purification. Studies have shown that the most common degradation products in the synthesis of polypeptides are deamidates, oxygenates, and hydrolysates. Among the various amino acids that make up a polypeptide, asparagine, glutamine and peptide chain C-segment amide are susceptible to deamidation reactions (especially at elevated pH and elevated temperatures).
The oxytocin [4-Glu ] impurity is a common impurity in the synthetic process of oxytocin, and can be used as an impurity reference substance in the quality detection of the oxytocin, so that the preparation of the oxytocin [4-Glu ] impurity with high purity has important significance for the quality control of the oxytocin.
At present, most of common purification methods for polypeptide drugs on the market adopt preparative high performance liquid chromatography, which is the most effective method for obtaining high-purity polypeptide target molecules. The general polypeptide medicine purification preparation process design is that target polypeptide is enriched by medium-low pressure chromatography and then refined by high pressure chromatography, but considering that the molecular weight of the target polypeptide oxytocin is about 1kDa, no proper molecular sieve gel column (the sample size is small, the flow rate is low, the treatment capacity is small, and the method is more suitable for desalting protein with the molecular weight of more than 10 kDa) or ultrafiltration membrane selection. And common separation methods in medium and low pressure chromatography include molecular sieve chromatography, ion exchange chromatography and hydrophobic interaction chromatography, the particle size of the filler used in the chromatography methods is usually different from dozens of micrometers to hundreds of micrometers, the size of the gap is mostly different from hundreds of nanometers, and the target polypeptide with high purity cannot be obtained. The concentrations of the oxytocin [4-Glu ] impurity crude products obtained by adopting solid phase synthesis and dilution cyclization are relatively dilute, and when a general reverse phase chromatographic column is adopted for purification, a large amount of organic waste liquid is generated only in the sample loading process, so that the treatment cost of hazardous waste is very high. Therefore, there is an urgent need to develop new economical and efficient processes suitable for purifying low concentrations of polypeptides and salts.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for refining oxytocin [4-Glu ] impurities, aiming at overcoming the defects of high treatment cost and uneconomic waste liquid caused by the generation of a large amount of organic waste liquid and large amount of dangerous waste liquid in the refining process of the oxytocin [4-Glu ] impurities in the prior art. Most of waste liquid generated in the purification process of the method for refining the oxytocin [4-Glu ] impurity is waste water which can be directly recycled through sewage treatment, and the method is economical and environment-friendly.
The invention solves the technical problems through the following technical scheme:
the invention provides a refining method of oxytocin [4-Glu ] impurities, which comprises the following steps:
adopting a high performance liquid phase reverse phase chromatography to carry out reverse phase enrichment, reverse phase salt conversion and reverse phase purification on the oxytocin [4-Glu ] impurity crude product solution in sequence;
the filler of the high performance liquid reverse phase chromatography is super water-resistant filler;
the reversed-phase enrichment, the reversed-phase salt conversion and the reversed-phase purification are all completed in the one-step reversed-phase elution process; the conditions of reversed phase enrichment, reversed phase salt conversion and reversed phase purification are as follows:
collecting the eluent with the retention time of 65-73 min to obtain oxytocin [4-Glu ] impurity solution;
the mobile phase A is acetic acid/water solution with the volume percentage of 0.005-0.1%, the mobile phase B is HAc/acetonitrile with the volume percentage of 0.005-0.1%, and the sample C1 is oxytocin [ 4-Glu%]The mobile phase C2 is 5-50 mM NH4Ac-NH4And (3) an OH aqueous solution, wherein the pH of the mobile phase C2 is 7.0-9.0, and the flow rate of the eluent is 80-100 ml/min.
In the invention, during 25-26 min, the eluent is changed from the sample C1 to the mobile phase C2; and during 40-41 min, replacing the eluent from the mobile phase C2 to the mobile phase A. According to the routine in the field, the time interval is not understood to be the limit of the elution condition, and the time can be adjusted according to the different types of the manufacturers of the high performance liquid chromatograph.
The crude oxytocin [4-Glu ] impurity solution is obtained by dissolving and diluting a solid-phase synthesized crude reduction type oxytocin [4-Glu ] impurity solution to obtain a crude reduction type oxytocin [4-Glu ] impurity solution, and oxidizing the crude reduction type oxytocin [4-Glu ] impurity solution.
The preparation method of the oxytocin [4-Glu ] impurity crude product solution comprises the following specific steps: taking Rink Amide MBHA resin as an initial raw material, taking amino acid protected by Fmoc as a monomer, taking HOBt/DIC as a condensing agent, and sequentially connecting the amino acid one by one; adding a peptide cutting reagent for peptide cutting, adding methyl tert-butyl ether for precipitation to obtain a crude product of the reduced oxytocin [4-Glu ] impurity; dissolving the crude reduced oxytocin [4-Glu ] impurity product with 50% acetic acid/water solution, and diluting with water to obtain crude reduced oxytocin [4-Glu ] impurity solution; and (2) adjusting the pH value of the reduced oxytocin [4-Glu ] impurity crude product solution to 7.0-9.0 by using an alkaline substance, adding 30% hydrogen peroxide for oxidation, and adding 0.5ml of 30% hydrogen peroxide into each gram of the reduced oxytocin [4-Glu ] impurity crude product to obtain an oxidized oxytocin [4-Glu ] impurity crude product solution, namely the oxytocin [4-Glu ] impurity crude product solution.
Wherein, the peptide cutting reagent can be conventional in the field, and preferably comprises the following components in a volume ratio of 90: 7.5: 2.5 TFA/TIS/H2O。
Wherein, the alkaline substance can be conventional in the field, and is preferably NaOH.
In the invention, the concentration of the crude reduced oxytocin [4-Glu ] impurity in the crude reduced oxytocin [4-Glu ] impurity solution is 0.1-4 mg/ml, preferably 0.5-2 mg/ml, such as 0.8mg/ml, 1mg/ml and 1.5 mg/ml.
In the present invention, the mobile phase a is preferably an acetic acid/water solution with a volume percentage of 0.02 to 0.05%.
The mobile phase B is preferably acetic acid/acetonitrile with the volume percentage of 0.02-0.05%.
The mobile phase C2 is preferably 10-20 mM NH4Ac-NH4An aqueous OH solution.
The pH of the mobile phase C2 is preferably 7.5-8.5.
In the invention, the HPLC purity of the oxytocin [4-Glu ] impurity in the oxytocin [4-Glu ] impurity crude product solution is 60-85%, preferably 70-80%.
Wherein, the oxytocin [4-Glu ] is]Oxytocin [4-Glu ] in impurity crude product solution]The structural formula of the impurity is
The solvent is an aqueous solution containing trifluoroacetic acid and acetic acid.
In the invention, the super waterproof filler is
ODS-AQ super water-resistant filler, preferably provided by Sovium nano-micro-technology GmbH
ODS-AQ super water-resistant filler. The aperture of the super water-resistant filler is preferably 7-10 nm, and the particle size of the super water-resistant filler is preferably 10 μm.
In the invention, the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
The reverse enrichment is an elution step (1), the reverse salt conversion is the elution steps (2) to (3), and specifically, the elution step (2) is performed by using the NH4Ac-NH4OH aqueous solution to remove oxytocin [4-Glu]And (3) removing ammonium ions in the elution step (2), and performing reverse phase purification to obtain the elution steps (4) and (5), wherein the elution step (4) is a process for removing weaker adsorbed impurities.
Wherein, the conversion rate of the eluent in the elution steps (4) and (5) is a process of uniform speed change, the uniform speed change rate in the elution step (4) is 2% of the mobile phase B/min, namely, 2% of the mobile phase B is increased on the basis of the original eluent every minute, and 2% of the mobile phase A is correspondingly reduced; the uniform speed change rate in the elution step (5) is 0.333 percent of the mobile phase B/min, namely, 0.333 percent of the mobile phase B is increased on the basis of the original eluent every minute, and 0.333 percent of the mobile phase A is correspondingly reduced at the same time.
And (3) changing the eluent from 80% of the mobile phase A + 20% of the mobile phase B to 50% of the mobile phase A + 50% of the mobile phase B within 83-84 min after the step (5) is finished. And 84-92 min, wherein the eluent is 50% of the mobile phase A + 50% of the mobile phase B. The aim of cleaning the chromatographic column is achieved by rapidly increasing the proportion of the organic phase.
The oxytocin [4-Glu ] impurity is a polypeptide substance, is unstable and easy to degrade under the condition of high pH, and particularly under the alkaline environment, the pH and the time of salt transfer elution are comprehensively considered, so that the damage and the loss of a sample in the salt transfer process are reduced.
In a preferred embodiment, Load is used&The Lock dynamic axial compression and static locking technology, the filler is
ODS-AQ super water-resistant filler with pore diameter of 10nm and particle diameter of 10 μm, packed to column bed pressure of 1000psi, using Varian chromatography packing system, 300g of the said filler in dry powder form
ODS-AQ super water-resistant filler, 600ml isopropanol, stirring and homogenizing, pouring into Load with inner diameter of 50mm&Lock4002 column preparation, compression ratio of 1.5:1, carrier gas N2The carrier gas pressure was adjusted to 1500psi oil gauge pressure and dynamically axially compressed to 25cm height of the bed as a preparative column for reverse phase enrichment, reverse phase salt conversion and reverse phase purification protocols.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the invention adopts an on-line enrichment method, utilizes the super-water-resistant performance and the adsorption performance of the filler, firstly adsorbs crude peptides in a polypeptide crude product solution to a stationary phase for enrichment, and hydrophobically combines the polypeptides and the reversed-phase filler.
(2) The method adopts on-line enrichment, can directly transform the mobile phase and then carry out gradient elution purification to obtain the final pure product, and is suitable for continuous production.
(3) The invention creatively uses the one-step method of reversed-phase adsorption enrichment, salt conversion and desalting to prepare the pure polypeptide product, optimizes the production process and is suitable for industrial continuous production.
(4) The invention designs the latest application of the super-waterproof filler, the mobile phases of the column balance stage, the sample loading enrichment stage and the salt conversion stage are saline water solutions, effluent liquid of the mobile phases is directly discharged to a sewage treatment station and can be recycled after simple treatment, and compared with the traditional preparation process, the invention greatly reduces the generation amount of hazardous waste and saves the environment.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following examples, the equipment conditions for detecting the purity of oxytocin [4-Glu ] impurity in the crude oxytocin [4-Glu ] impurity and the purified product solution by HPLC method are as follows:
the instrument comprises the following steps: agilent 1200 high performance liquid chromatograph
Separating the column: waters Xbridge-C18, 4.6X 150mm, 5 μm
Mobile phase: a is acetonitrile water solution with the volume percentage of 50 percent, B is 0.02M KH2PO4pH 3.0 aqueous solution, flow Rate1.0ml/min, the detection wavelength is 220nm, the detection is carried out at room temperature, the elution gradient is shown in the following table, and the percentage is volume percentage.
In the following embodiments, the crude oxytocin [4-Glu ] impurity solution is obtained by dissolving and diluting a solid-phase synthesized crude reduced oxytocin [4-Glu ] impurity to obtain a crude reduced oxytocin [4-Glu ] impurity solution, and oxidizing the crude reduced oxytocin [4-Glu ] impurity solution.
The oxytocin [4-Glu]The specific preparation steps of the crude impurity solution are as follows: taking Rink Amide MBHA resin as an initial raw material, taking amino acid protected by Fmoc as a monomer, taking HOBt/DIC as a condensing agent, and sequentially connecting the amino acid one by one; adding peptide cutting reagent to cut peptide, adding methyl tert-butyl ether to precipitate to obtain reduced oxytocin [4-Glu]Crude product of impurities; the reduction type oxytocin [4-Glu ] is reacted]Dissolving the crude impurity product with 50% acetic acid/water solution, and diluting with water to obtain reduced oxytocin [ 4-Glu%]A crude solution of impurities; reducing oxytocin [4-Glu ] by using alkaline substance]Adjusting the pH of the crude impurity solution to 7.0-9.0, adding 30% hydrogen peroxide solution, and oxidizing per gram of reduced oxytocin [ 4-Glu%]Adding 0.5ml of 30% hydrogen peroxide into the crude product of the impurity to obtain oxidized oxytocin [4-Glu ]]The impurity crude product solution is the oxytocin [4-Glu]Crude solution of impurities. Wherein, the peptide cutting reagent is 90: 7.5: 2.5 TFA/TIS/H2And O. Wherein, the alkaline substance is NaOH.
Example 1 preparation of column packing with 50mm ID Load & Lock4002
Application of Load&The Lock dynamic axial compression and static locking technology, the filler is
ODS-AQ, pore size 10nm, particle size 10 μm, packed to bed pressure 1000psi, using a Varian chromatography packing system, 300g of said as a dry powder
ODS-AQ super water-resistant filler, 600ml isopropanol, stirring and homogenizing, pouring into Load with inner diameter of 50mm&Lock4002 column preparation, compression ratio of 1.5:1, carrier gas N2The carrier gas pressure was adjusted to 1500psi oil gauge pressure and dynamically axially compressed to 25cm height of the bed as a preparative column for reverse phase enrichment, reverse phase salt conversion and reverse phase purification protocols.
Example 2 reverse enrichment, reverse salt conversion and reverse purification of oxytocin [4-Glu ] impurity crude solution
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: preparative column Load of example 1&Lock4002 50×250mm，
ODS-AQ particle size is 10 μm, pore diameter is 10nm
Oxytocin [4-Glu]The crude product of impurities has the structural formula
Oxytocin [4-Glu]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid, reduced oxytocin [4-Glu]Reduced oxytocin [4-Glu ] in impurity crude product solution]The concentration of the crude impurity was 1 mg/ml.
Mobile phase a was 0.02% by volume acetic acid/water solution, mobile phase B was 0.02% by volume acetic acid/acetonitrile, sample C1 was oxytocin [ 4-Glu%]Crude solution of impurities, said oxytocin [4-Glu determined according to HPLC method]The HPLC purity of the impurity was 75.23%, and the mobile phase C2 was 10mM NH4Ac-NH4The pH of the aqueous OH solution, mobile phase C2, was 7.5.
The reverse phase enrichment, reverse phase salt conversion and reverse phase purification conditions of this example are as follows: the flow rate was 100ml/min, the detection wavelength was 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
Collecting the eluent with the retention time of 65-73 min to obtain the oxytocin [4-Glu ] impurity solution. The purity of oxytocin [4-Glu ] impurity HPLC determined according to HPLC method was 99.42%.
Example 3 reverse enrichment, reverse salt conversion and reverse purification of oxytocin [4-Glu ] impurity crude solution
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: preparative column Load of example 1&Lock4002 50×250mm，
ODS-AQ particle size is 10 μm, pore diameter is 10 nm.
Oxytocin [4-Glu]The structural formula of the impurity is
Oxytocin [4-Glu]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid, reduced oxytocin [4-Glu]Reduced oxytocin [4-Glu ] in impurity crude product solution]The concentration of the crude impurity was 1.5 mg/ml.
Mobile phase a was 0.05% by volume acetic acid/water solution, mobile phase B was 0.05% by volume acetic acid/acetonitrile, sample C1 was oxytocin [ 4-Glu%]Crude solution of impurities, said oxytocin [4-Glu determined according to HPLC method]Purity of impurity HPLC 70.65%, mobile phase C2 20mM NH4Ac-NH4The pH of the aqueous OH solution, mobile phase C2, was 8.5.
The reverse phase enrichment, reverse phase salt conversion and reverse phase purification conditions of this example are as follows: the flow rate was 100ml/min, the detection wavelength was 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
Collecting the eluent with the retention time of 65-73 min to obtain the oxytocin [4-Glu ] impurity solution. The oxytocin [4-Glu ] impurity HPLC purity as determined by HPLC method is 99.46%.
Example 4 reverse enrichment, reverse salt conversion and reverse purification of oxytocin [4-Glu ] impurity crude solution
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: preparative column Load of example 1&Lock4002 50×250mm，
ODS-AQ particle size is 10 μm, pore diameter is 10nm
Oxytocin [4-Glu]The crude product of impurities has the structural formula
Oxytocin [4-Glu]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid, reduced oxytocin [4-Glu]Reduced oxytocin [4-Glu ] in impurity crude product solution]The concentration of the crude impurity was 0.8 mg/ml.
The mobile phase A is 0.05% by volume acetic acid/water solution, the mobile phase B is 0.05% by volume acetic acid/acetonitrile, and the sample C1 is oxytocin [4-Glu]Crude solution of impurities, said oxytocin [4-Glu determined according to HPLC method]Purity of impurity HPLC 75.16%, mobile phase C2 20mM NH4Ac-NH4The pH of the aqueous OH solution, mobile phase C2, was 7.5.
The reverse phase enrichment, reverse phase salt conversion and reverse phase purification conditions of this example are as follows: the flow rate was 100ml/min, the detection wavelength was 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
Collecting the eluent with the retention time of 65-73 min to obtain the oxytocin [4-Glu ] impurity solution. The purity of the oxytocin [4-Glu ] impurity HPLC determined according to HPLC method was 99.25%.
Example 5 Mass Spectrometry detection of oxytocin [4-Glu ] impurities
Oxytocin [4-Glu ] impurities obtained in examples 2, 3 and 4 were measured by Waters micromass ZQ single quadrupole electrospray mass spectrometry (ESI-MS) under the following test conditions: performing mass spectrometry by using an electrospray ionization (ESI) source in a positive ionization mode, wherein the ionization voltage of a capillary tube is 3.0kV, and the sampling taper hole voltage is 35 kV; the ion source temperature is 115 ℃, the desolventizing temperature is 350 ℃, the desolventizing nitrogen flow rate is 700L/h, the cone hole back flushing nitrogen flow rate is 50L/h, and the sweep range of the four-level rod is 50.0-1500 m/z.
The detection result is as follows: molecular ion Peak [ M + H]+Mass to charge ratio (M/z) of 1008.43, main ion fragment peak [ M +2H]2+The mass-to-charge ratios (m/z) are 504.71, which all conform to the theoretical value (oxytocin [4-Glu ]]The relative molecular mass of the impurity is 1008.18).
Claims (10)
1. A refining method of oxytocin [4-Glu ] impurities is characterized by comprising the following steps: adopting a high performance liquid phase reverse phase chromatography to carry out reverse phase enrichment, reverse phase salt conversion and reverse phase purification on the oxytocin [4-Glu ] impurity crude product solution in sequence;
the oxytocin [4-Glu]The structural formula of the impurity is
；
The filler of the high performance liquid reverse phase chromatography is super water-resistant filler; the super water-resistant filler is UniSil
ODS-AQ super water-resistant filler;
the reversed-phase enrichment, the reversed-phase salt conversion and the reversed-phase purification are all completed in the one-step reversed-phase elution process; the conditions of reversed phase enrichment, reversed phase salt conversion and reversed phase purification are as follows:
collecting the eluent with the retention time of 65-73 min to obtain oxytocin [4-Glu ] impurity solution;
the mobile phase A is acetic acid/water solution with the volume percentage of 0.005-0.1%, the mobile phase B is acetic acid/acetonitrile solution with the volume percentage of 0.005-0.1%, and the sample C1 is oxytocin [4-Glu]The mobile phase C2 is 5-50 mM NH4Ac-NH4And (3) an OH aqueous solution, wherein the pH of the mobile phase C2 is 7.0-9.0, and the flow rate of the eluent is 80-100 ml/min.
2. The method for purifying oxytocin [4-Glu ] impurities according to claim 1, characterized in that: changing the eluent from the sample C1 to the mobile phase C2 in the period of 25-26 min; and during 40-41 min, replacing the eluent from the mobile phase C2 to the mobile phase A.
3. The method for purifying oxytocin [4-Glu ] impurities according to claim 1, characterized in that: the crude oxytocin [4-Glu ] impurity solution is obtained by dissolving and diluting a solid-phase synthesized crude reduction type oxytocin [4-Glu ] impurity solution to obtain a crude reduction type oxytocin [4-Glu ] impurity solution, and oxidizing the crude reduction type oxytocin [4-Glu ] impurity solution.
4. The method for purifying oxytocin [4-Glu ] impurities according to claim 3, characterized in that: the concentration of the reduction type oxytocin [4-Glu ] impurity crude product in the reduction type oxytocin [4-Glu ] impurity crude product solution is 0.1-4 mg/ml.
5. The method for purifying oxytocin [4-Glu ] impurities according to claim 4, characterized in that: the concentration of the reduction type oxytocin [4-Glu ] impurity crude product in the reduction type oxytocin [4-Glu ] impurity crude product solution is 0.5-2 mg/ml.
6. The method for purifying oxytocin [4-Glu ] impurities according to claim 1, characterized in that:
the mobile phase A is an acetic acid/water solution with the volume percentage of 0.02-0.05%;
and/or the mobile phase B is an acetic acid/acetonitrile solution with the volume percentage of 0.02-0.05%;
and/or the mobile phase C2 is 10-20 mM NH4Ac-NH4An aqueous OH solution;
and/or the pH of the mobile phase C2 is 7.5-8.5;
and/or the HPLC purity of the oxytocin [4-Glu ] impurity in the oxytocin [4-Glu ] impurity crude product solution is 60-85%.
7. The method for purifying oxytocin [4-Glu ] impurities according to claim 6, characterized in that: the HPLC purity of the oxytocin [4-Glu ] impurity in the oxytocin [4-Glu ] impurity crude product solution is 70-80%.
8. The method for purifying oxytocin [4-Glu ] impurities according to claim 1, characterized in that: in the oxytocin [4-Glu ] impurity crude product solution, a solvent is an aqueous solution containing trifluoroacetic acid and acetic acid.
9. The method for purifying oxytocin [4-Glu ] impurities according to claim 1, characterized in that: the aperture of the super water-resistant filler is 7-10 nm, and the particle size of the super water-resistant filler is 10 microns.
10. The method for purifying oxytocin [4-Glu ] impurities according to claim 1, characterized in that: the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910376583.0A CN110078797B (en) | 2019-05-07 | 2019-05-07 | Method for refining oxytocin [4-Glu ] impurity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910376583.0A CN110078797B (en) | 2019-05-07 | 2019-05-07 | Method for refining oxytocin [4-Glu ] impurity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110078797A CN110078797A (en) | 2019-08-02 |
| CN110078797B true CN110078797B (en) | 2021-04-13 |
Family
ID=67419067
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910376583.0A Active CN110078797B (en) | 2019-05-07 | 2019-05-07 | Method for refining oxytocin [4-Glu ] impurity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110078797B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011018745A1 (en) * | 2009-08-11 | 2011-02-17 | Biocon Limited | Chromatographic processes and purified compounds thereof |
| CN106188218A (en) * | 2016-07-14 | 2016-12-07 | 江苏诺泰生物制药股份有限公司 | A kind of method improving polypeptide raw material drug stabilisation |
| CN106478780A (en) * | 2017-01-03 | 2017-03-08 | 上海上药第生化药业有限公司 | A kind of preparation method of oxytocin [4 Glu] |
-
2019
- 2019-05-07 CN CN201910376583.0A patent/CN110078797B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011018745A1 (en) * | 2009-08-11 | 2011-02-17 | Biocon Limited | Chromatographic processes and purified compounds thereof |
| CN106188218A (en) * | 2016-07-14 | 2016-12-07 | 江苏诺泰生物制药股份有限公司 | A kind of method improving polypeptide raw material drug stabilisation |
| CN106478780A (en) * | 2017-01-03 | 2017-03-08 | 上海上药第生化药业有限公司 | A kind of preparation method of oxytocin [4 Glu] |
Non-Patent Citations (1)
| Title |
|---|
| 醋酸奥曲肽的合成研究;刘作家;《中国优秀硕士学位论文全文数据库》;20041215(第4期);E079-9 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110078797A (en) | 2019-08-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110016072B (en) | Method for refining oxytocin | |
| CN106478780B (en) | Preparation method of oxytocin [4-Glu ] | |
| CN109929010B (en) | Method for refining vasopressin | |
| CN106518978B (en) | Preparation method of vasopressin [4-Glu,5-Asp ] | |
| CN106749541B (en) | Preparation method of vasopressin [5-Asp ] | |
| CN103980351A (en) | Method for preparing vasopressin and vasopressin tannate | |
| CN106518977B (en) | Preparation method of oxytocin [5-Asp ] | |
| CN105131079B (en) | A kind of purification process of desmopressin acetate | |
| CN109942686B (en) | Purification method of vasopressin acetylated impurities | |
| CN110078797B (en) | Method for refining oxytocin [4-Glu ] impurity | |
| CN110041406B (en) | Method for refining oxytocin [ + Gly ] impurity | |
| CN110066319B (en) | Method for refining oxytocin acetylation impurities | |
| CN106518976B (en) | Preparation method of oxytocin [4-Glu,5-Asp ] | |
| CN110041405B (en) | Method for refining oxytocin [5-Asp ] impurity | |
| CN110028556B (en) | Method for refining oxytocin impurity [ -NH2] | |
| CN110078796B (en) | Method for refining oxytocin [4-Glu,5-Asp ] impurity | |
| CN110016070B (en) | Method for refining impurities of vasopressin [ + Gly ] | |
| CN110016071B (en) | Method for refining vasopressin [4-Glu ] impurity | |
| CN109929011B (en) | Method for refining vasopressin [5-Asp ] impurity | |
| CN110003313B (en) | Vasopressin [ -NH ]2]Method for purifying impurities | |
| CN110078795B (en) | Method for refining vasopressin [4-Glu,5-Asp ] impurity | |
| CN109942678B (en) | Refining method of octreotide | |
| CN106749540B (en) | Preparation method of vasopressin [4-Glu ] | |
| WO2021129016A1 (en) | Method for desalting polypeptides | |
| CN106749528B (en) | Preparation method of octreotide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |