CN110003313B - Vasopressin [ -NH ]2]Method for purifying impurities - Google Patents
Vasopressin [ -NH ]2]Method for purifying impurities Download PDFInfo
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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Abstract
The invention discloses a vasopressin [ -NH ]2]A method for purifying impurities. Vasopressin [ -NH ]2]The purification method of impurities comprises the following steps: adopts high performance liquid reverse phase chromatography to convert vasopressin to [ -NH ]2]Sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the impurity crude product solution; the filler of the high performance liquid phase reverse phase chromatography is super water-resistant filler; reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification are all completed in a one-step reversed-phase elution process. Vasopressin [ -NH ] of the invention2]Most of waste liquid generated in the purification process of the impurity refining method is waste water, can be recycled by simple treatment of a sewage station, and is economic and environment-friendly.
Description
Technical Field
The invention relates to a vasopressin [ -NH ]2]A method for purifying 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).
Wherein vasopressin [ -NH ]2]The impurities are common impurities in the synthesis process of the vasopressin, and can be used as impurity reference substances in the quality detection of the vasopressin, so that the high-purity vasopressin [ -NH ] is prepared2]The impurities have important significance on the 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 process design for purifying and preparing polypeptide substances is to enrich target polypeptide by medium-low pressure chromatography and then refine by high pressure chromatography, but considering target polypeptide vasopressin [ -NH ]2]The molecular weight of the impurities is about 1kDa, and no proper molecular sieve gel column (the sample weight is small, the flow rate is low, the treatment capacity is small, and the method is more suitable for desalting proteins with the molecular weight of more than 10 kDa) or ultrafiltration membrane selection is available. 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. Vasopressin [ -NH obtained by solid phase synthesis plus dilution cyclization2]The concentrations of impurity crude product solutions are relatively dilute, a common reversed-phase chromatographic column is adopted for purification, a large amount of organic waste liquid is generated only in the processes of sample loading and salt conversion, the organic waste liquid cannot be directly discharged or can be recycled through simple treatment of a sewage station, especially, the low-concentration sample is purified, the amount of the waste liquid is larger, and the treatment cost of hazardous waste is very high. Therefore, there is an urgent need to develop new polypeptides and salts suitable for purification at low concentrationsIs an economical and efficient process.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the vasopressin [ -NH ] in the prior art2]A great amount of organic waste liquid is generated in the refining process of the impurities, the amount of hazardous waste liquid is large, the treatment cost of the waste liquid is high, and the defects of uneconomic performance are caused, thereby providing the vasopressin [ -NH ]2]A method for purifying impurities. The refined vasopressin of the invention [ -NH ]2]Most of waste liquid generated in the purification process of the impurity method is waste water, can be recycled by simple treatment of a sewage station, and is economic and environment-friendly.
The invention solves the technical problems through the following technical scheme:
the present invention provides one kind of vasopressin [ -NH ]2]A method for purifying impurities, comprising the steps of:
adopts high performance liquid reverse phase chromatography to convert vasopressin to [ -NH ]2]Sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the impurity crude product solution;
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:
the mobile phase A is acetic acid/water solution with the volume percentage of 0.005-0.1 percent, the mobile phase B is acetic acid/acetonitrile with the volume percentage of 0.005-0.1 percent, and the sample C1 is the vasopressin [ -NH ]2]The mobile phase C2 is 5-50 mM NH4Ac-NH4The pH value of the mobile phase C2 is 7.0-9.0, and the flow rate of the eluent is 80-100 mL/min; collecting the eluent with retention time of 85-94 min to obtain vasopressin [ -NH ]2]An impurity solution.
In the invention, the eluent is changed from the sample C1 to the mobile phase C2 within 30-31 min. And during 50-51 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 vasopressin [ -NH ]2]The preparation method of the crude impurity solution comprises the following steps:
(1) preparing reduced vasopressin [ -NH ] by solid phase synthesis2]Crude product of impurities;
(2) the reduced vasopressin [ -NH ]2]Dissolving and diluting the crude impurity product to obtain reduced vasopressin < -NH2]A crude solution of impurities;
(3) the reduced vasopressin [ -NH ]2]Oxidizing the crude solution of impurities to obtain the vasopressin < -NH2]Crude solution of impurities.
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 peptide cutting reagent to cut peptide, adding methyl tert-butyl ether to make precipitation to obtain reduced vasopressin [ -NH ]2]Crude product of impurities.
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 may be conventional in the art, and preferably the reduced form of vasopressin [ -NH ] is treated with an alkaline material2]And adjusting the pH value of the impurity crude product solution to 7.0-9.0, and adding 30% hydrogen peroxide by volume percentage to perform an oxidation process. The dosage of the hydrogen peroxide is 0.5mL/1g reduced vasopressin [ -NH ]2]Crude product of impurities.
Wherein, the alkaline substance can be conventional in the field, and is preferably NaOH.
In the present invention, the reduced vasopressin [ -NH ]2]Reduced vasopressin [ -NH ] in solution of crude impurity2]The concentration of the crude product of impurities is 0.1-4 mg/mL, preferably 0.5-2 mg/mL, for example, 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 present invention, the vasopressin [ -NH ]2]The vasopressin [ -NH ] in the crude solution of impurities2]The HPLC purity of the impurities is 60-85%, preferably 70-80%.
The vasopressin [ -NH ]2]The vasopressin [ -NH ] in the crude solution of impurities2]The structure of the impurity is
In the present invention, the vasopressin [ -NH ]2]The solvent in the crude impurity solution 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 of Sozhou Nami 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.
And (3) after the elution step (5) is finished for 100-101 min, replacing 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. And 101-113 min, wherein the eluent is 50% of the mobile phase A and 50% of the mobile phase B. The aim of cleaning the chromatographic column is achieved by rapidly increasing the proportion of the mobile phase B.
The reversed phase enrichment is the elution step (1), and the reversed phase salt conversion is the elution steps (2) to (3). Specifically, the elution step (2) is carried out by using the NH4Ac-NH4Removal of said vasopressin [ -NH ] by aqueous OH solution2]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.
The vasopressin [ -NH ]2]The impurity is a polypeptide substance which is unstable and easy to degrade under the condition of high pH, and particularly under the alkali environment, the invention 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 10nm of pore diameter and 10 mu m of particle size300g of said dry powder, loaded to a bed pressure of 1000psi, using a Varian chromatographic loading system
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 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 super-water-resistant filler, and firstly adopts the vasopressin [ -NH ]2]Vasopressin [ -NH ] in crude solution of impurities2]Adsorbing the coarse impurity onto the stationary phase for enriching, and adding vasopressin-NH2]The crude impurities and the reversed phase filler are combined in a hydrophobic way.
(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 inverse enrichment, the inverse salt conversion and the inverse purification to prepare the vasopressin [ -NH ] by a one-step method2]The impurity is pure, the production process is optimized, and the method is suitable for industrial continuous production.
(4) The latest application of the super-waterproof filler is designed, the eluents in the column balance stage, the reversed-phase enrichment stage and the reversed-phase salt conversion stage are aqueous solutions, the environment is protected, no pollution is caused, the effluent liquid is directly discharged to a sewage treatment station, the effluent liquid can be recycled after simple treatment, the generation amount of hazardous waste is greatly reduced compared with the traditional preparation process, and the environment is saved.
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, HPLC method was used to detect vasopressin [ -NH ]2]Vasopressin [ -NH ] in crude impurity and purified product solutions2]The equipment conditions for impurity purity were 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, vasopressin [ -NH ]2]The preparation method of the crude impurity solution comprises the following steps: (1) preparing reduced vasopressin [ -NH ] by solid phase synthesis2]Crude product of impurities; (2) the reduced vasopressin [ -NH ]2]Dissolving and diluting the crude impurity product to obtain reduced vasopressin < -NH2]A crude solution of impurities; (3) the reduced vasopressin [ -NH ]2]Oxidizing the crude solution of impurities to obtain the vasopressin < -NH2]Crude solution of impurities.
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 peptide cutting reagent to cut peptide, adding methyl tert-butyl ether to make precipitation to obtain reduced vasopressin [ -NH ]2]Crude product of impurities. The peptide cutting reagent is prepared from the following components in a volume ratio of 90: 7.5: 2.5 TFA/TIS/H2And O. The dissolution is carried out by using 50 percent by volume of acetic acid/water solution. The dilution is water dilution. The oxidation is to make the reduced vasopressin [ -NH ] by alkaline substance2]Adjusting the pH value of the impurity crude product solution to 7.0-9.0, and adding 30% hydrogen peroxide by volumeAnd (4) an oxidation process. The dosage of the hydrogen peroxide is 0.5mL/1g reduced vasopressin [ -NH ]2]Crude product of impurities. 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 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 vasopressin [ -NH ]2]Reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification of impurity crude product 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&Lock 400250X 250mm and the filler is
ODS-AQ particle size is 10 μm, pore diameter is 10 nm.
Vasopressin [ -NH ]2]The structural formula of the impurity is
Reduced vasopressin [ -NH ]2]Reduced vasopressin [ -NH ] in solution of crude impurity2]The concentration of the crude product of the impurity is 1mg/mL, and the concentration of vasopressin [ -NH ]2]The solvent in the crude impurity solution is a solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is in volume percent0.02% acetic acid/water solution, mobile phase B0.02% acetic acid/acetonitrile by volume, sample C1 vasopressin [ -NH ]2]Crude solution of impurity, HPLC purity according to HPLC method 77.11%, mobile phase C2 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 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 retention time of 85-94 min to obtain vasopressin [ -NH ]2]An impurity solution. The HPLC purity according to the HPLC method was 99.19%.
Example 3 vasopressin [ -NH ]2]Reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification of impurity crude product 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&Lock 400250X 250mm and the filler is
ODS-AQ particle size is 10 μm, pore diameter is 10 nm.
Vasopressin [ -NH ]2]The structural formula of the impurity is
Reduced vasopressin [ -NH ]2]Reduced vasopressin [ -NH ] in solution of crude impurity2]The concentration of the crude product of impurities is 1.5mg/mL, vasopressin [ -NH ]2]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
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 vasopressin [ -NH2]Crude solution of impurities having an HPLC purity of72.63% and 20mM NH in mobile phase C24Ac-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 retention time of 85-94 min to obtain vasopressin [ -NH ]2]An impurity solution. The HPLC purity according to the HPLC method was 99.52%.
Example 4 vasopressin [ -NH ]2]Reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification of impurity crude product 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&Lock 400250X 250mm and the filler is
ODS-AQ particle size is 10 μm, pore diameter is 10nm
Vasopressin [ -NH ]2]The structural formula of the impurity is
Reduced vasopressin [ -NH ]2]Reduced vasopressin [ -NH ] in solution of crude impurity2]The concentration of the crude product of impurities is 0.8mg/mL, vasopressin [ -NH ]2]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
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 vasopressin [ -NH2]Crude solution of impurity, HPLC purity according to HPLC method 74.86%, 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 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 retention time of 85-94 min to obtain vasopressin [ -NH ]2]An impurity solution. The HPLC purity according to the HPLC method was 99.22%.
Example 5 vasopressin [ -NH ]2]Mass spectrometric detection of impurities
The vasopressin [ -NH ] obtained in examples 2, 3 and 4 was measured by Waters micromass ZQ single quadrupole electrospray mass spectrometry (ESI-MS)2]Impurities, the test conditions were: 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 1085.42, main ion fragment peak [ M +2H]2+The mass-to-charge ratios (m/z) are 543.21, both of which correspond to the theoretical values (vasopressin [ -NH ]2]The relative molecular mass of the impurity is 1085.25).
Claims (12)
1. Vasopressin [ -NH ]2]A method for purifying impurities, characterized by comprising the steps of: adopts high performance liquid reverse phase chromatography to convert vasopressin to [ -NH ]2]Sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the impurity crude product solution;
the vasopressin [ -NH ]2]The structure of the impurity is
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:
the mobile phase A is acetic acid/water solution with the volume percentage of 0.005-0.1 percent, the mobile phase B is acetic acid/acetonitrile with the volume percentage of 0.005-0.1 percent, and the sample C1 is the vasopressin [ -NH ]2]The mobile phase C2 is 5-50 mM NH4Ac-NH4The pH value of the mobile phase C2 is 7.0-9.0, and the flow rate of the eluent is 80-100 mL/min; collecting the eluent with retention time of 85-94 min to obtain vasopressin [ -NH ]2]An impurity solution.
2. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: changing the eluent from the sample C1 to the mobile phase C2 within 30-31 min; and during 50-51 min, replacing the eluent from the mobile phase C2 to the mobile phase A.
3. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: the conversion rate of the eluent in the elution steps (4) and (5) is a process with uniform change.
4. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: the vasopressin [ -NH ]2]The preparation method of the crude impurity solution comprises the following steps:
(1) preparing reduced vasopressin [ -NH ] by solid phase synthesis2]Crude product of impurities;
(2) the reduced vasopressin [ -NH ]2]Dissolving crude product of impuritiesDiluting to obtain reduced vasopressin [ -NH ]2]A crude solution of impurities;
(3) the reduced vasopressin [ -NH ]2]Oxidizing the crude solution of impurities to obtain the vasopressin < -NH2]Crude solution of impurities.
5. The vasopressin [ -NH ] of claim 42]A method for purifying impurities, characterized by comprising: the reduced vasopressin [ -NH ]2]Reduced vasopressin [ -NH ] in solution of crude impurity2]The concentration of the crude product of the impurity is 0.1-4 mg/mL.
6. The vasopressin [ -NH ] of claim 52]A method for purifying impurities, characterized by comprising: the reduced vasopressin [ -NH ]2]Reduced vasopressin [ -NH ] in solution of crude impurity2]The concentration of the crude product of impurities is 0.5-2 mg/mL.
7. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: 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, said vasopressin [ -NH ]2]The vasopressin [ -NH ] in the crude solution of impurities2]The HPLC purity of the impurities is 60-85%.
8. The vasopressin [ -NH ] of claim 72]A method for purifying impurities, characterized by comprising: the vasopressin [ -NH ]2]The vasopressin [ -NH ] in the crude solution of impurities2]The HPLC purity of the impurities is 70-80%.
9. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: the vasopressin [ -NH ]2]In the crude impurity solution, the solvent is water solution containing trifluoroacetic acid and acetic acid.
10. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: 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.
11. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: the super water-resistant filler is UniSil
ODS-AQ super water-resistant filler.
12. The vasopressin [ -NH ] of claim 12]A method for purifying impurities, characterized by comprising: the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
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| CN106749541A (en) * | 2017-01-03 | 2017-05-31 | 上海上药第生化药业有限公司 | A kind of preparation method of pitressin [5 Asp] |
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| CN103102395A (en) * | 2012-12-18 | 2013-05-15 | 深圳翰宇药业股份有限公司 | Preparation method of desmopressin acetate |
| CN106518978A (en) * | 2017-01-03 | 2017-03-22 | 上海上药第生化药业有限公司 | Preparation method of pitressin [4-Glu,5-Asp] |
| CN106699851A (en) * | 2017-01-03 | 2017-05-24 | 上海上药第生化药业有限公司 | Method for preparing vasopressin deamidization impurities |
| CN106749541A (en) * | 2017-01-03 | 2017-05-31 | 上海上药第生化药业有限公司 | A kind of preparation method of pitressin [5 Asp] |
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