CN110066319B - Method for refining oxytocin acetylation impurities - Google Patents
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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 refining method of oxytocin acetylation impurities, which comprises the following steps: sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the oxytocin acetylation impurity crude product solution by adopting a high performance liquid reversed-phase chromatography; 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 oxytocin acetylation impurities has the advantages that most of waste liquid generated in the purification process is waste water, the waste water can be recycled through simple treatment of a sewage station, and the method is economical and environment-friendly.
Description
Technical Field
The invention relates to a refining method of oxytocin acetylation 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 acetylation impurities are common impurities in the synthesis process of oxytocin, and the impurities can be used as impurity reference substances in the quality detection of the oxytocin, so that the preparation of the oxytocin acetylation impurities with high purity has important significance on the quality control of the oxytocin.
At present, most of common purification methods of polypeptide medicines 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 crude oxytocin acetylation impurity solutions obtained by adopting solid phase synthesis and dilution cyclization are relatively dilute, when a general reverse 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 transferring, 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 waste liquid amount is larger, and 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 acetylated impurities, aiming at overcoming the defects of high treatment cost and low economy of waste liquid caused by the fact that a large amount of organic waste liquid is generated in the refining process of the oxytocin acetylated impurities and the amount of dangerous waste liquid is large in the prior art. Most of waste liquid generated in the purification process of the method for refining the oxytocin acetylation impurities 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 acetylation impurities, which comprises the following steps:
sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the oxytocin acetylation impurity crude product solution by adopting a high performance liquid reversed-phase chromatography;
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 81-90 min to obtain an oxytocin acetylation impurity solution;
the mobile phase A is acetic acid/water solution with volume percentage of 0.005-0.1%, the mobile phase B is acetic acid/acetonitrile with volume percentage of 0.005-0.1%, the sample C1 is the oxytocin acetylation impurity crude product solution, and 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 isIs 80 to 100 ml/min.
In the invention, the eluent is changed from the sample C1 to the mobile phase C2 within 40-41 min; and during 48-49 min, replacing the eluent with the mobile phase A from the mobile phase C2. 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 acetylation impurity solution is prepared by dissolving and diluting a solid-phase synthesized reduction-type oxytocin acetylation impurity crude product to obtain a reduction-type oxytocin acetylation impurity crude product solution, and oxidizing the reduction-type oxytocin acetylation impurity crude product solution.
The preparation method of the oxytocin acetylation 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 reduced oxytocin acetylation impurities; dissolving the reduced oxytocin acetylated impurity crude product by using 50% by volume of acetic acid/water solution, and diluting by using water to obtain a reduced oxytocin acetylated impurity crude product solution; adjusting the pH value of the reduced oxytocin acetylation 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 acetylation impurity crude product to obtain an oxidized oxytocin acetylation impurity crude product solution, namely the oxytocin acetylation 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 reduced oxytocin acetylated impurity crude product in the reduced oxytocin acetylated impurity crude product solution 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 invention, the HPLC purity of the oxytocin acetylation impurities in the oxytocin acetylation impurity crude product solution is 60-85%, preferably 70-80%.
Wherein the structural formula of the oxytocin acetylation impurities in the oxytocin acetylation impurity crude product solution is shown in the specification
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 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-NH4The process of removing trifluoroacetate in the oxytocin acetylation impurity crude product by using OH aqueous solution, wherein the elution step (3) is to remove the trifluoroacetate in the oxytocin acetylation impurity crude productThe process of eluting ammonium ions in step (2), the reverse phase purification is the elution steps (4) and (5), wherein the elution step (4) is a process of removing weak 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) after the step (5) is finished, for 92-93 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. 93-105 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 acetylation impurities are polypeptide substances which are unstable and easy to degrade under the condition of high pH, and particularly in an 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 preparation column, compression ratio1.5:1, 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 acetylation impurities in the crude oxytocin acetylation impurities and in the purified product solution by the 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 water solution, flow rate of 1.0ml/min, detection wavelength of 220nm, room temperature detection, and elution gradient shown in the following table, the percentage is volume percentage.
| Step of elution | Elution time | Eluent |
| 1 | 0～15min | 35％A+65％B |
| 2 | 15～25min | 35％A+65％B→100％A |
| 3 | 25～28min | 100％A |
| 4 | 28～28.1min | 100％A→35％A+65％B |
| 5 | 28.1～32min | 35％A+65％B |
In the following embodiments, the crude oxytocin acetylated impurity solution is obtained by dissolving and diluting a reduced oxytocin acetylated impurity crude product synthesized by a solid phase to obtain a reduced oxytocin acetylated impurity crude product solution, and oxidizing the reduced oxytocin acetylated impurity crude product solution.
The preparation method of the oxytocin acetylation 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 reduced oxytocin acetylation impurities; dissolving the reduced oxytocin acetylated impurity crude product by using 50% by volume of acetic acid/water solution, and diluting by using water to obtain a reduced oxytocin acetylated impurity crude product solution; adjusting the pH value of the reduced oxytocin acetylation 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 acetylation impurity crude product to obtain an oxidized oxytocin acetylation impurity crude product solution, namely the oxytocin acetylation impurity crude product solution. 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-AQThe super water-resistant filler is stirred and homogenized by 600ml of isopropanol, and then poured into a Load with the 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 phase enrichment, reverse phase salt conversion and reverse phase purification apparatus of oxytocin acetylation impurity crude product solution: 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
The structural formula of the oxytocin acetylation impurities is
The solvent in the crude oxytocin acetylation impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid, and the concentration of the crude reduced oxytocin acetylation impurity in the crude reduced oxytocin acetylation impurity solution is 1 mg/ml.
The mobile phase A is acetic acid/water solution with the volume percentage of 0.02 percent, the mobile phase B is acetic acid/acetonitrile with the volume percentage of 0.02 percent, the sample C1 is oxytocin acetylation impurity crude product solution, the HPLC purity of the oxytocin acetylation impurity is 77.40 percent according to the HPLC method, and the mobile phase C2 is 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 81-90 min to obtain the oxytocin acetylation impurity solution. The purity of the oxytocin acetylation impurity HPLC was 99.68% as determined by HPLC method.
Example 3 reverse phase enrichment, reverse phase salt conversion and reverse phase purification apparatus of oxytocin acetylation impurity crude product solution: 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
The structural formula of the oxytocin acetylation impurities is
The solvent in the crude oxytocin acetylation impurity solution is water solution containing trifluoroacetic acid and acetic acid, and the concentration of the crude reduced oxytocin acetylation impurity in the crude reduced oxytocin acetylation impurity solution is 1.5 mg/ml.
The mobile phase A is acetic acid/water solution with the volume percentage of 0.05 percent, the mobile phase B is acetic acid/acetonitrile with the volume percentage of 0.05 percent, the sample C1 is oxytocin acetylation impurity crude product solution, the purity of the oxytocin acetylation impurity HPLC determined by an HPLC method is 73.13 percent, and the mobile phase C2 is 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 81-90 min to obtain the oxytocin acetylation impurity solution. The purity of the oxytocin acetylation impurity by HPLC method was 99.25%.
Example 4 reverse phase enrichment, reverse phase salt conversion and reverse phase purification apparatus of oxytocin acetylation impurity crude solution: 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
The structural formula of the oxytocin acetylation impurities is
The solvent in the crude oxytocin acetylation impurity solution is water solution containing trifluoroacetic acid and acetic acid, and the concentration of the crude reduced oxytocin acetylation impurity in the crude reduced oxytocin acetylation impurity solution is 0.8 mg/ml.
The mobile phase A is 0.05 percent of acetic acid/water solution by volume percentage, the mobile phase B is 0.05 percent of acetic acid/acetonitrile by volume percentage, the sample C1 is a crude oxytocin acetylation impurity solution, the HPLC purity of the oxytocin acetylation impurity is 72.86 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 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 81-90 min to obtain the oxytocin acetylation impurity solution. The HPLC purity of the oxytocin acetylation impurity determined according to HPLC method was 99.25%.
Example 5 Mass Spectrometry detection of oxytocin acetylation impurities
The oxytocin acetylation 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 1049.50, main ion fragment peak [ M +2H]2+The mass to charge ratios (m/z) were 525.26, all in line with the theoretical values (relative molecular mass of oxytocin acetylation impurity was 1049.26).
Claims (10)
1. A refining method of oxytocin acetylation impurities is characterized by comprising the following steps: sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the oxytocin acetylation impurity crude product solution by adopting a high performance liquid reversed-phase chromatography;
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 81-90 min to obtain an oxytocin acetylation impurity solution;
the mobile phase A is an acetic acid/water solution with the volume percentage of 0.005-0.1%, the mobile phase B is an acetic acid/acetonitrile solution with the volume percentage of 0.005-0.1%, the sample C1 is the oxytocin acetylation impurity crude product solution, and 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 80-100ml/min；
The structural formula of the oxytocin acetylation impurities in the oxytocin acetylation impurity crude product solution is shown in the specification
。
2. A process for the purification of an acetylated oxytocin impurity according to claim 1, characterised in that: changing the eluent from the sample C1 to the mobile phase C2 within 40-41 min; and during 48-49 min, replacing the eluent with the mobile phase A from the mobile phase C2.
3. A process for the purification of an acetylated oxytocin impurity according to claim 1, characterised in that: the crude oxytocin acetylation impurity solution is prepared by dissolving and diluting a solid-phase synthesized reduction-type oxytocin acetylation impurity crude product to obtain a reduction-type oxytocin acetylation impurity crude product solution, and oxidizing the reduction-type oxytocin acetylation impurity crude product solution.
4. A process for the purification of an acetylated oxytocin impurity according to claim 3, characterised in that: the concentration of the reduced oxytocin acetylated impurity crude product in the reduced oxytocin acetylated impurity crude product solution is 0.1-4 mg/ml.
5. The method of refining oxytocin acetylation impurities according to claim 4, wherein: the concentration of the reduced oxytocin acetylated impurity crude product in the reduced oxytocin acetylated impurity crude product solution is 0.5-2 mg/ml.
6. A process for the purification of an acetylated oxytocin impurity according to claim 1, characterised 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 acetylation impurities in the oxytocin acetylation impurity crude product solution is 60-85%.
7. The method of refining oxytocin acetylation impurities according to claim 6, wherein: the HPLC purity of the oxytocin acetylation impurities in the oxytocin acetylation impurity crude product solution is 70-80%.
8. A process for the purification of an acetylated oxytocin impurity according to claim 1, characterised in that: the solvent in the oxytocin acetylation impurity crude product solution is water solution containing trifluoroacetic acid and acetic acid.
9. A process for the purification of an acetylated oxytocin impurity according to claim 1, characterised 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. A process for the purification of an acetylated oxytocin impurity according to claim 1, characterised in that: the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
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| 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 |
| CN106674332A (en) * | 2017-01-03 | 2017-05-17 | 上海上药第生化药业有限公司 | Preparation method of oxytocin |
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| 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 |
| CN106674332A (en) * | 2017-01-03 | 2017-05-17 | 上海上药第生化药业有限公司 | Preparation method of oxytocin |
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| 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 * |
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