CN109942686B - Purification method of vasopressin acetylated impurities - Google Patents
Purification method of vasopressin acetylated impurities Download PDFInfo
- Publication number
- CN109942686B CN109942686B CN201910371418.6A CN201910371418A CN109942686B CN 109942686 B CN109942686 B CN 109942686B CN 201910371418 A CN201910371418 A CN 201910371418A CN 109942686 B CN109942686 B CN 109942686B
- Authority
- CN
- China
- Prior art keywords
- vasopressin
- acetylation
- phase
- impurity
- purification
- 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
Landscapes
- Peptides Or Proteins (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for refining acetylated vasopressin impurities. The purification method of the vasopressin acetylation impurities comprises the following steps: performing reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the crude vasopressin acetylation impurity solution by adopting a high performance liquid reversed-phase chromatography 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 the acetylated vasopressin 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 method for refining acetylated vasopressin impurities.
Background
Vasopressin is a synthetic polypeptide consisting of nine amino acid residues and has the chemical structure
Theoretical molecular weight of vasopressin 1084.24. Belongs to the neurohypophysis hormone, also called vasopressin or vasopressin, and has two receptors, V1 and V2. V1 is mainly distributed on the cell membrane of vascular smooth muscle, and acts through receptor-G protein-second messenger pathway to make blood vessel contract and raise blood pressure. V2 epithelial cells in the distal tubule and collecting duct of kidney, and physiological dosage can promote reabsorption of water by the distal tubule and collecting duct of kidney, and exert antidiuretic effect.
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 examination of the purity thereof is one of the important bases for ensuring the safety and effectiveness of the drug, and the content of the purity examination is somewhat different depending on the properties and characteristics of each drug, but basically involves examination and study of respective "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 related substances, and 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 can be very similar to the properties of the drug, 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 method comprises the following steps of preparing a high-purity vasopressin acetylation impurity, wherein the vasopressin acetylation impurity is a common impurity in a synthesis process of vasopressin, and can be used as an impurity reference substance in quality detection of vasopressin, so that the preparation of the high-purity vasopressin acetylation impurity has important significance on quality control of the vasopressin.
At present, most of common purification methods of polypeptide substances 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 target polypeptide vasopressin acetylated impurities 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 is provided. 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 solution concentration of the crude product of the acetylation impurities of the vasopressin obtained by adopting solid phase synthesis and dilution cyclization is relatively dilute, a common reversed phase chromatographic column is adopted for purification, a large amount of organic waste liquid is generated in the sample loading process, the organic waste liquid cannot be directly discharged or can be recycled by simple treatment of a sewage station, especially, the purification treatment of a sample with low concentration has larger waste liquid amount and high treatment cost of hazardous waste. 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 the acetylation impurities of vasopressin, aiming at overcoming the defects of high treatment cost and low economy of 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 acetylation impurities of vasopressin in the prior art. The method for refining the acetylation impurities of the vasopressin has the advantages that most of waste liquid generated in the process of purifying a target product is waste water, can be directly recycled through sewage treatment, and is economical and environment-friendly.
The invention solves the technical problems through the following technical scheme:
the invention provides a method for refining acetylated vasopressin impurities, which comprises the following steps: performing reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the crude vasopressin acetylation impurity solution by adopting a high performance liquid reversed-phase chromatography 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 84-92 min to obtain a vasopressin acetylation 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 with the volume percentage of 0.005-0.1%, the sample C1 is the crude vasopressin acetylation impurity solution, and 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 35-36 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.
In the invention, the process of uniform speed change in the elution step (4) is that 2% of the mobile phase B is increased on the basis of the original eluent every minute, and simultaneously 2% of the mobile phase A is correspondingly reduced; in the step (5), the uniform speed change process is that every minute, on the basis of the original eluent, 0.333% of the mobile phase B is added, and meanwhile, 0.333% of the mobile phase A is correspondingly reduced.
The crude vasopressin acetylation impurity solution is prepared by dissolving and diluting a reduced vasopressin acetylation impurity crude product prepared by a solid-phase synthesis method into a reduced vasopressin acetylation impurity crude product solution, and oxidizing the reduced vasopressin acetylation impurity crude product solution.
The specific preparation steps of the crude vasopressin acetylation 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 a peptide cutting reagent for peptide cutting, adding methyl tert-butyl ether for precipitation to obtain a reduced vasopressin acetylated impurity crude product; dissolving and diluting the reduced vasopressin acetylated impurity crude product to obtain a reduced vasopressin acetylated impurity crude product solution; adjusting the pH value of the reduced vasopressin acetylated impurity crude product solution to 7.0-9.0 by using an alkaline substance, adding 30% hydrogen peroxide to carry out the oxidation process, and adding 0.5ml of 30% hydrogen peroxide to each gram of the reduced vasopressin acetylated impurity crude product to obtain an oxidized vasopressin acetylated impurity crude product solution, namely the vasopressin acetylated 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。
The dissolution may be conventional in the art and is preferably carried out with 50% by volume acetic acid/water solution.
The dilution may be conventional in the art, preferably with water.
The oxidation process can be conventional in the field, and preferably, the pH value of the reduced vasopressin acetylation impurity crude product solution is adjusted to 7.0-9.0 by using an alkaline substance, and hydrogen peroxide with the volume percentage of 30% is added for oxidation. The dosage of the hydrogen peroxide is 0.5mL/1g of reduced vasopressin acetylated impurity crude product.
Wherein, the alkaline substance can be conventional in the field, and is preferably NaOH.
In the invention, the concentration of the reduced form vasopressin acetylated impurity crude product 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 invention, the HPLC purity of the crude vasopressin acetylation impurities in the crude vasopressin acetylation impurities solution is 60-85%, preferably 70-80%.
The structural formula of the vasopressin acetylation impurities in the crude vasopressin acetylation impurity solution is shown in the specification
In the invention, the solvent in the crude vasopressin acetylation impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
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.
The detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
In the invention, 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;
the super water-resistant filler is
ODS-AQ super water-resistant filler, preferably Suzhou Na microOf science and technology Ltd
ODS-AQ super water-resistant filler.
In addition, the step (6) is 93-94 min, wherein 80% of mobile phase A + 20% of mobile phase B → 50% of mobile phase A + 50% of mobile phase B, and the step (7) is 94-109 min, wherein 50% of mobile phase A + 50% of mobile phase B. The aim of cleaning the chromatographic column is achieved by rapidly increasing the proportion of the organic phase.
Wherein the reverse enrichment is the elution step (1), the reverse salt conversion is the elution steps (2) to (3), and specifically, the elution step (2) is performed with the NH4Ac-NH4And (3) removing the trifluoroacetate in the crude vasopressin acetylation impurity solution by using an OH aqueous solution, wherein the elution step (3) is a process for removing ammonium ions in the elution step (2), and the reversed-phase purification is the elution steps (4) to (5), wherein the elution step (4) is a process for removing weaker adsorption impurities.
The acetylation impurities of the vasopressin are polypeptide substances which are unstable and easy to degrade under the condition of high pH, and particularly under the alkaline environment, the method comprehensively considers the pH and the time of the eluent of the reverse phase salt conversion so as to ensure that the damage and the loss of the sample in the reverse phase salt conversion 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 is stirred and homogenized by 600mL of isopropanol, and then Load with the inner diameter of 50mm is poured into the mixture&Lock4002 column preparation, compression ratio of 1.5:1, carrier gas N2The carrier gas pressure is adjusted so that the oil pressure gauge pressure is1500psi, dynamic axial compression to a bed height of 25cm, as a preparative column for reverse phase enrichment, reverse phase salt conversion and reverse phase purification schemes.
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 latest application of the super-waterproof filler is designed, the mobile phase in the column balance stage, the sample loading enrichment stage and the salt transferring stage can be aqueous solution, the method is environment-friendly and pollution-free, the effluent liquid of the method is directly discharged to a sewage treatment station and can be recycled after simple treatment, compared with the traditional preparation process, the method greatly reduces the generation amount of hazardous waste, and saves the environment.
(2) 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.
(3) 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.
(4) 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.
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 HPLC analysis of the purity of the crude and purified vasopressin acetylation impurities in solution are as follows:
the instrument comprises the following steps: agilent1260 high performance liquid chromatograph
A chromatographic column: waters XBridgeC184.6X 250mm, 5 μm
Mobile phase: a is 0.1% by volume aqueous TFA, B is 0.1% by volume TFA-50% aqueous acetonitrile (TFA is trifluoroacetic acid)
The flow rate is 1.0mL/min, the detection wavelength is 210nm, and the column temperature is as follows: the elution gradient is shown in the table below at 25 ℃ and the percentages are by volume.
| Step of elution | Elution time | Eluent |
| 1 | 0～2min | 95％A+5％B |
| 2 | 2～12min | 95％A+5％B→85％A+15％B |
| 3 | 12～22min | 85％A+15％B |
| 4 | 22～30min | 85％A+15％B→77％A+23％B |
| 5 | 30～30.1min | 77％A+23％B→50％A+50％B |
| 6 | 30.1～35min | 50％A+50％B |
In the following examples, the preparation of a crude solution of an acetylation impurity of vasopressin comprises: (1) preparing a crude product of reduced type vasopressin acetylation impurities by adopting a solid phase synthesis method; (2) dissolving and diluting the reduced vasopressin acetylated impurity crude product to obtain a reduced vasopressin acetylated impurity crude product solution; (3) and oxidizing the reduced crude vasopressin acetylation impurity solution to obtain the crude vasopressin acetylation impurity solution.
The solid phase synthesis method comprises the following 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, and obtaining a crude product of the reduced vasopressin acetylated impurities. Wherein, the volume ratio of the peptide cutting reagent is 90: 7.5: 2.5 TFA/TIS/H2And O. The dissolution was carried out with 50% by volume acetic acid/water solution. The dilution is water dilution. The oxidation process is to adjust the pH of the reduced vasopressin acetylated impurity crude product solution to 7.0-9.0 by using alkaline substances, and add 30 volume percent of hydrogen peroxide for oxidation. The dosage of the hydrogen peroxide is 0.5mL/1g of reduced vasopressin acetylated impurity crude product. 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, Varian chromatography packing system, 300g dry powder as described
ODS-AQ super water-resistant filler is stirred and homogenized by 600mL of isopropanol, and then Load with the inner diameter of 50mm is poured&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 phase enrichment, reverse phase salt conversion and reverse phase purification of crude solution of acetylated impurities of vasopressin
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: preparative column Load of example 1&Lock 400250X 250mm and the filler is
ODS-AQ, particle size 10 μm, pore size 10 nm.
The structural formula of the acetylation impurities of vasopressin is shown in the specification
The concentration of the reduced type crude vasopressin acetylation impurity solution is 1mg/mL, and the solvent of the crude vasopressin acetylation impurity solution is water solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is 0.02 percent by volume of acetic acid/water solution, the mobile phase B is 0.02 percent by volume of acetic acid/acetonitrile, the sample C1 is a crude solution of the acetylation impurities of vasopressin, the HPLC purity of the acetylation impurities of vasopressin is 78.36 percent determined by an HPLC method, and the mobile phase C2 is 10mM of 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 is 100mL/min, the detection wavelength is 220nm, the purification elution gradient is shown in the following table, and the percentage is volume percentage;
collecting the eluent with the retention time of 84-92 min to obtain the vasopressin acetylation impurity solution. The HPLC purity of the vasopressin acetylation impurity determined according to HPLC method was 99.68%.
EXAMPLE 3 reverse phase enrichment, reverse phase salt conversion and reverse phase purification of crude solution of acetylated impurities of vasopressin
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: preparative column Load of example 1&Lock 400250X 250mm and the filler is
ODS-AQ, particle size 10 μm, pore size 10 nm.
The structural formula of the acetylation impurities of vasopressin is shown in the specification
The concentration of the reduced type crude vasopressin acetylation impurity solution is 1.5mg/mL, and the solvent of the crude vasopressin acetylation impurity solution is water solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is 0.05 percent by volume of acetic acid/water solution, the mobile phase B is 0.05 percent by volume of acetic acid/acetonitrile, the sample C1 is crude solution of the acetylation impurities of the vasopressin, the HPLC purity of the acetylation impurities of the vasopressin is 72.13 percent determined by an HPLC method, and the mobile phase C2 is 20mM of 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 is 100mL/min, the detection wavelength is 220nm, the purification elution gradient is shown in the following table, and the percentage is volume percentage;
collecting the eluent with the retention time of 84-92 min to obtain the vasopressin acetylation impurity solution. The HPLC purity of the vasopressin acetylation impurity determined according to HPLC method was 99.25%.
Example 4 reverse phase enrichment, reverse phase salt conversion and reverse phase purification of a solution of crude vasopressin acetylation impurities
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: preparative column Load of example 1&Lock 400250X 250mm and the filler is
ODS-AQ, particle size 10 μm, pore size 10 nm.
The structural formula of the acetylation impurities of vasopressin is shown in the specification
The concentration of the reduced type crude vasopressin acetylation impurity solution is 0.8mg/mL, and the solvent of the crude vasopressin acetylation impurity solution is water solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is 0.05 percent by volume of acetic acid/water solution, the mobile phase B is 0.05 percent by volume of acetic acid/acetonitrile, the sample C1 is crude solution of the acetylation impurities of the vasopressin, the HPLC purity of the acetylation impurities of the vasopressin is 75.46 percent determined by an HPLC method, and the mobile phase C2 is 20mM of 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 is 100mL/min, the detection wavelength is 220nm, the purification elution gradient is shown in the following table, and the percentage is volume percentage;
collecting the eluent with the retention time of 84-92 min to obtain the vasopressin acetylation impurity solution. The HPLC purity of the vasopressin acetylation impurity determined according to HPLC method was 99.30%.
Example 5 Mass Spectrometry detection of Presser acetylation impurities
The acetylated vasopressin impurities obtained in examples 2, 3 and 4 were determined by means of 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 1128.43, main ion fragment peak [ M +2H]2+The mass to charge ratio (m/z) was 564.73, all in line with the theoretical value (relative molecular mass of the vasopressin acetylation impurity was 1128.34).
Claims (12)
1. A method for refining a vasopressin acetylation impurity, which is characterized by comprising the following steps: performing reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the crude vasopressin acetylation impurity solution by adopting a high performance liquid reversed-phase chromatography in sequence;
the structural formula of the vasopressin acetylation impurities in the crude vasopressin acetylation impurity solution is shown in the specification
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 84-92 min to obtain a vasopressin acetylation 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 with the volume percentage of 0.005-0.1%, the sample C1 is the crude product solution of the acetylation impurities of the vasopressin, and the sample C1 is the crude product solution of the acetylation impurities of the vasopressinThe 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. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: changing the eluent from the sample C1 to the mobile phase C2 in the period of 25-26 min; and during 35-36 min, replacing the eluent from the mobile phase C2 to the mobile phase A.
3. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: in the elution steps (4) and (5), the conversion rate of the eluent is a process with uniform change.
4. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: the crude vasopressin acetylation impurity solution is prepared by dissolving and diluting a reduced vasopressin acetylation impurity crude product prepared by a solid-phase synthesis method into a reduced vasopressin acetylation impurity crude product solution, and oxidizing the reduced vasopressin acetylation impurity crude product solution.
5. A process for the purification of a vasopressin acetylation impurity as claimed in claim 4, wherein: the concentration of the reduced vasopressin acetylated impurity crude product solution is 0.1-4 mg/mL.
6. A process for the purification of a vasopressin acetylation impurity as claimed in claim 5, wherein: the concentration of the reduced vasopressin acetylated impurity crude product solution is 0.5-2 mg/mL.
7. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: the HPLC purity of the vasopressin acetylation impurities in the crude vasopressin acetylation impurities solution is 60-85%.
8. A process for the purification of a vasopressin acetylation impurity as claimed in claim 7, wherein: the HPLC purity of the vasopressin acetylation impurities in the crude vasopressin acetylation impurities solution is 70-80%.
9. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: in the crude vasopressin acetylation impurity solution, a solvent is an aqueous solution containing trifluoroacetic acid and acetic acid.
10. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: 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 acetic acid/acetonitrile 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 detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
11. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: 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.
12. A process for the purification of a vasopressin acetylation impurity as claimed in claim 1, wherein: the super water-resistant filler is
ODS-AQ super water-resistant filler.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910371418.6A CN109942686B (en) | 2019-05-06 | 2019-05-06 | Purification method of vasopressin acetylated impurities |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910371418.6A CN109942686B (en) | 2019-05-06 | 2019-05-06 | Purification method of vasopressin acetylated impurities |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109942686A CN109942686A (en) | 2019-06-28 |
| CN109942686B true CN109942686B (en) | 2021-06-25 |
Family
ID=67016973
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910371418.6A Active CN109942686B (en) | 2019-05-06 | 2019-05-06 | Purification method of vasopressin acetylated impurities |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109942686B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113698456B (en) * | 2021-05-31 | 2024-05-28 | 海南双成药业股份有限公司 | Purification method of arginin vasopressin |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2237356T3 (en) * | 2003-04-07 | 2007-04-01 | Novetide Ltd. | PROCEDURE FOR THE PRODUCTION OF CYCLIC PEPTIDES. |
| WO2006041945A2 (en) * | 2004-10-04 | 2006-04-20 | Novetide, Ltd. | A counterion exchange process for peptides |
| MX346473B (en) * | 2009-08-11 | 2017-03-22 | Biocon Ltd | Chromatographic processes and purified compounds thereof. |
| CN106518978B (en) * | 2017-01-03 | 2019-12-31 | 上海上药第一生化药业有限公司 | Preparation method of vasopressin [4-Glu,5-Asp ] |
| CN106699851B (en) * | 2017-01-03 | 2019-10-15 | 上海上药第一生化药业有限公司 | A kind of preparation method of pitressin deamidation impurity |
| CN106749541B (en) * | 2017-01-03 | 2020-02-18 | 上海上药第一生化药业有限公司 | Preparation method of vasopressin [5-Asp ] |
| CN106518975B (en) * | 2017-01-03 | 2019-10-15 | 上海上药第一生化药业有限公司 | A kind of preparation method of pitressin |
-
2019
- 2019-05-06 CN CN201910371418.6A patent/CN109942686B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109942686A (en) | 2019-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109929010B (en) | Method for refining vasopressin | |
| CN103980351A (en) | Method for preparing vasopressin and vasopressin tannate | |
| CN106518978B (en) | Preparation method of vasopressin [4-Glu,5-Asp ] | |
| CN106749541B (en) | Preparation method of vasopressin [5-Asp ] | |
| CN106478780B (en) | Preparation method of oxytocin [4-Glu ] | |
| CN110016072B (en) | Method for refining oxytocin | |
| CN109942686B (en) | Purification method of vasopressin acetylated impurities | |
| CN106518977B (en) | Preparation method of oxytocin [5-Asp ] | |
| 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 | |
| CN109438561A (en) | A kind of purification process of Triptorelin | |
| CN110078797B (en) | Method for refining oxytocin [4-Glu ] impurity | |
| CN110041406B (en) | Method for refining oxytocin [ + Gly ] impurity | |
| CN109942678B (en) | Refining method of octreotide | |
| CN110066319B (en) | Method for refining oxytocin acetylation impurities | |
| CN103965291A (en) | Method for preparing octreotide and octreotide acetate | |
| CN110041405B (en) | Method for refining oxytocin [5-Asp ] impurity | |
| CN110078796B (en) | Method for refining oxytocin [4-Glu,5-Asp ] impurity | |
| CN110028556B (en) | Method for refining oxytocin impurity [ -NH2] | |
| CN106749540B (en) | Preparation method of vasopressin [4-Glu ] | |
| CN106518976B (en) | Preparation method of oxytocin [4-Glu,5-Asp ] | |
| CN109748954B (en) | Purification method of degarelix |
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 |