CN110028556B - Method for refining oxytocin impurity [ -NH2] - Google Patents
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Abstract
The invention discloses oxytocin [ -NH ]2]A method for purifying impurities, the method comprising the steps of: adopts high performance liquid phase reverse phase chromatography to convert oxytocin to NH2]Sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the impurity crude product solution; oxytocin [ NH ]2]The crude solution of impurity is reduced oxytocin [ -NH synthesized by solid phase2]Oxidizing the crude impurity solution to obtain the impurity solution; the filler of the high performance liquid phase reverse phase chromatography is super water-resistant filler. The invention uses one-step method of on-line reverse phase enrichment, reverse phase salt conversion and reverse phase purification to prepare pure polypeptide product oxytocin-NH2]The removal rate of impurities in the crude product of the impurities is higher, and oxytocin < - > -NH is obtained2]The purity of the pure impurity product is high, the mobile phases of the column balance stage, the sample loading enrichment stage and the salt conversion stage are aqueous solutions, the method is environment-friendly and pollution-free, and effluent waste liquid can be directly subjected to sewage treatment and recycled.
Description
Technical Field
The invention relates to oxytocin [ -NH ]2]A method for purifying 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 placenta reserve function, namely 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 of the synthesized polypeptide, such as deletion peptides, broken peptides, oxidized peptides, products of disulfide bond exchange, and the like, 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 amides are susceptible to deamidation reactions, particularly at elevated pH and elevated temperatures.
At present, most of common purification methods for polypeptide drugs on the market adopt preparative high performance liquid chromatography, which is the most effective method for obtaining high-purity polypeptide target molecules. The general polypeptide medicine purification preparation process design is that target polypeptide is enriched by medium-low pressure chromatography and then refined by high pressure chromatography, but considering that the molecular weight of the target polypeptide oxytocin is about 1kDa, no proper molecular sieve gel column (the sample size is small, the flow rate is low, the treatment capacity is small, and the method is more suitable for desalting protein with the molecular weight of more than 10 kDa) or ultrafiltration membrane selection. And common separation methods in medium and low pressure chromatography include molecular sieve chromatography, ion exchange chromatography and hydrophobic interaction chromatography, the particle size of the filler used in the chromatography methods is usually different from dozens of micrometers to hundreds of micrometers, the size of the gap is mostly different from hundreds of nanometers, and the target polypeptide with high purity cannot be obtained. The concentration of the oxytocin crude product solution obtained by adopting solid phase synthesis and dilution cyclization is relatively dilute, and when a general reverse phase chromatographic column is adopted for purification, a large amount of organic waste liquid is generated only in the sample loading process, so that the treatment cost of hazardous waste is very high. There is still a lack of an efficient method for preparing polypeptide salt drug substances, and there is still a great need to develop new economical and efficient processes suitable for purifying low concentrations of polypeptides and salts.
Chinese patent document CN 106749539a discloses a preparation method of oxytocin deamidation. The preparation method comprises the following steps: sequentially carrying out reversed-phase cyclization, reversed-phase purification and reversed-phase desalting on the oxytocin deamidation precursor crude product solution by adopting a high performance liquid reversed-phase chromatography; the filler of the high performance liquid reverse phase chromatography is C18; the oxytocin deamidate precursor crude product is an oxytocin deamidate precursor crude product containing two free sulfydryls. In the preparation process, a reduction-type oxytocin deamidation precursor crude product is used as an initial raw material, the removal rate of impurities in the crude product is low, and NaOH is used as an alkaline substance in a mobile phase, so that the pH is not controlled well, and the stability of a product is possibly influenced. In addition, the method needs a large amount of mobile phase containing organic solvent when the cyclization is carried out on a chromatographic column, a large amount of organic hazardous waste liquid is generated in the steps of purification and salt conversion, and the treatment cost of the subsequent waste liquid is high and the subsequent waste liquid is difficult to recycle.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defect of refining oxytocin < - > -NH in the prior art2]Oxytocin [ NH ] as an impurity2]The removal rate of impurities in the impurity crude product solution is low, the produced hazardous waste liquid has large amount and is organic hazardous waste liquid, the treatment cost of the waste liquid is high, and the defect of being uneconomical is overcome, thereby providing the oxytocin [ -NH ]2]A method for purifying impurities. The refined oxytocin of the invention [ -NH ]2]Most of waste liquid generated in the process of purifying target products by the impurity method is waste water which can be directly recycled by sewage treatment, and the method is economical and environment-friendly, and oxytocin [ -NH ]2]The removal rate of impurities in the crude impurity solution is high.
The invention solves the technical problems through the following technical scheme:
the invention provides oxytocin [ -NH ]2]A method for purifying impurities, comprising the steps of:
adopts high performance liquid phase reverse phase chromatography to convert oxytocin to NH2]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 reverse phase enrichment, reverse phase salt conversion and reverse phase purification are all completed in one-step reverse phase elution process, and the conditions of the reverse phase enrichment, the reverse phase salt conversion and the reverse phase purification are as follows:
wherein, the mobile phase A is acetic acid/water 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 oxytocin [ -NH2]Crude impurity solution with a mobile phase C2 of 5-50 mM NH4Ac-NH4The pH value of the mobile phase C2 is 7.0-9.0, and the flow rate is 80-100 ml/min;
collecting the eluent with retention time of 80-89 min to obtain oxytocin [ -NH ]2]An impurity solution.
In the invention, oxytocin [ -NH ] is used2]The crude solution of impurity is reduced oxytocin [ -NH synthesized by solid phase2]The crude product of the impurity is obtained by dissolution, dilution and oxidation.
In the invention, oxytocin [ -NH ] is used2]The specific preparation steps of the crude impurity solution are as follows: taking Rink Amide MBHA resin as an initial raw material, taking amino acid protected by Fmoc as a monomer, taking HOBt/DIC as a condensing agent, and sequentially connecting the amino acid one by one; adding peptide cutting reagent to cut peptide, adding methyl tert-butyl ether to make precipitation to obtain reduced oxytocin [ -NH ]2]Crude product of impurities; reducing oxytocin [ -NH ]2]Dissolving the crude product with 50% acetic acid/water, and adding waterDiluting to obtain reduced oxytocin [ -NH ]2]A crude solution of impurities; reducing oxytocin [ -NH ] with alkaline substance2]Adjusting the pH value of the crude impurity solution to 7.0-9.0, adding 30% hydrogen peroxide for oxidation, wherein each gram of reduced oxytocin [ -NH ]2]0.5ml of 30% hydrogen peroxide is added into the crude product of the impurity to obtain oxidized oxytocin [ -NH ]2]The impurity crude product solution is oxytocin [ -NH ]2]Crude solution of impurities.
Wherein 50% acetic acid/water can sufficiently dissolve the reduced oxytocin < - > -NH2]Crude product of impurities.
Wherein, the reduction type oxytocin [ -NH ]2]The concentration of the crude impurity solution is 0.1-4 mg/ml, preferably 0.5-2 mg/ml.
Wherein the peptide cutting reagent is conventional in the field, and preferably TFA/TIS/H with the volume ratio of 90:7.5:2.52O。
Wherein, the alkaline substance is conventional in the field, and is preferably NaOH.
In the present invention, oxytocin [ -NH ] is described2]Oxytocin < - > -NH in crude impurity solution2]The structural formula of the impurity is
The oxytocin [ -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.
In the invention, the aperture of the super-waterproof filler is 7-10 nm, and the particle size is 10 μm.
In a preferred embodiment, Load is used&The Lock dynamic axial compression and static locking technology adopts the packing material of
ODS-AQ super water-resistant filler with 10nm of pore diameter and 10 mu of particle diameterm, packed to bed pressure 1000psi, using a Varian chromatography packing system, 300g 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 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.
In the invention, the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
In the invention, 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 oxytocin [ -NH ]2]The HPLC purity of the crude impurity solution is 60-85%, preferably 70-85%, more preferably 70-80%;
and/or collecting the eluent with the retention time of 83-86 min to obtain oxytocin [ -NH ]2]An impurity solution.
In the invention, the eluent is completely changed from a sample C1 to a mobile phase C2 within 45-46 min; and completely replacing the eluent from the mobile phase C2 to the mobile phase A within 51-52 min. 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, within 97-98 min after the step (5) is finished, the eluent is reduced from 80% of the mobile phase A to 50% of the mobile phase A at a constant speed, the mobile phase B is correspondingly increased to 50% at a constant speed, and the elution of 50% of the mobile phase A and 50% of the mobile phase B is kept within 98-110 min, so that the aim of cleaning the chromatographic column is fulfilled.
Wherein the reversed phase enrichment is an elution step (1), the reversed phase salt conversion is steps (2) to (3), and specifically, the step (2) is to use weak base NH4Ac-NH4OH removal of oxytocin [ -NH ]2]And (3) removing ammonium ions in the step (2), and performing reverse phase purification to obtain steps (4) and (5), wherein the step (4) is a process for removing weaker adsorbed impurities.
Wherein, the mobile phase of column balance, the enrichment of going up the appearance and changeing the salt stage is the aqueous solution that contains the salt, and the environmental protection is pollution-free, and the effluent waste liquid can directly carry out simple sewage treatment and recycle, very big reduction the cost that the danger waste liquid was handled.
In the invention, 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% 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 decreased at the same time; the uniform speed change rate in the elution step (5) is 0.333% B/min, namely, 0.333% of the mobile phase B is increased on the basis of the original eluent every minute, and 0.333% of the mobile phase A is correspondingly reduced at the same time.
Oxytocin [ NH ]2]The impurities 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 salt transfer elution so as to ensure that the damage and the loss of the sample in the salt transfer process are reduced.
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 to adsorb the crude polypeptide product onto the stationary phase for enrichment, and the polypeptide and the reversed-phase filler are combined in a hydrophobic manner. After on-line enrichment, the mobile phase can be directly transformed and then gradient elution purification is carried out to obtain the final pure product, and the method is suitable for continuous production.
(2) 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. Oxytocin [ NH ]2]The removal rate of impurities in the crude impurity solution is higher.
(3) 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 production amount of dangerous waste liquid 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.
The water-resistant fillers used in the examples were obtained from Suzhou Nami microbial science and technology Ltd
ODS-AQ super water-resistant filler with a pore diameter of 10nm and a particle size of 10 μm.
HPLC method for detecting oxytocin [ -NH ]2]Purity of crude impurity and purified product solution:
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 examples, oxytocin [ -NH ] is described2]The crude solution of impurity is reduced oxytocin [ -NH synthesized by solid phase2]The crude product of the impurity is obtained by dissolution, dilution and oxidation. The solid phase synthesis method comprises the following steps: by RinkTaking 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 oxytocin [ -NH ]2]Crude product of impurities. The peptide cutting reagent is TFA/TIS/H with the volume ratio of 90:7.5:2.52And 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 use alkaline substance to make the reduction type oxytocin [ -NH ]2]Adjusting the pH value of the impurity crude product solution to 7.0-9.0, and adding 30% hydrogen peroxide by volume percentage for oxidation. The dosage of the hydrogen peroxide is 0.5mL/1g reduced oxytocin [ -NH ]2]Crude product of impurities. The alkaline substance is NaOH.
Example 1 preparation of column packing with 50mm inner diameter L & L4002:
application of Load&The Lock dynamic axial compression and static locking technology adopts the packing material of
ODS-AQ with aperture of 10nm and particle diameter of 10 μm is packed to column bed pressure of 1000psi, and stirred and homogenized by Varian chromatography packing system, 300g dry powder packing and 600ml isopropanol, and poured into L with inner diameter of 50mm&L4002 column preparation, compression ratio 1.5:1, carrier gas N2The carrier gas pressure was adjusted to 1500psi oil gauge pressure and dynamic axial compression to 25cm column bed height was used as a preparative column for reverse phase enrichment, reverse phase salt conversion and reverse phase purification protocols.
Example 2 oxytocin [ -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: EXAMPLE 1 self-contained preparative column Load&Lock4002 50×250mm，
ODS-AQ 10μm 10nm
Oxytocin [ NH ]2]The structural formula of the impurity is
Reduced oxytocin [ NH ]2]Reduced oxytocin [ NH ] in impurity crude product solution2]The concentration of the crude product of impurities is 1.0mg/ml, oxytocin [ -NH ]2]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
Mobile phase a was 0.02% acetic acid/water by volume, mobile phase B was 0.02% acetic acid/acetonitrile by volume, sample C1 was oxytocin [ -NH2]Crude solution of impurities, oxytocin [ -NH ] determined according to HPLC method2]The HPLC purity of the crude solution of the impurity was 72.23%, 10mM NH in mobile phase C24Ac-NH4OH pH7.5 aqueous solution.
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 was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
Collecting the eluent with retention time of 80-89 min to obtain oxytocin [ -NH ]2]An impurity solution. oxytocin-NH measured according to HPLC method2]The HPLC purity of the impurity was 99.32%. Oxytocin [ NH ]2]The removal rate of impurities in the crude impurity solution is 27.09%; collecting the eluent with retention time of 83-86 min to obtain oxytocin-NH2]The HPLC purity of the impurity was 99.83%.
In the embodiment, the eluent in the steps (1) to (3) is aqueous solution, is environment-friendly and pollution-free, the effluent liquid of the eluent is directly discharged to a sewage treatment station, and can be recycled after simple treatment, so that the generation amount of hazardous waste liquid is greatly reduced, and the environment is saved compared with the traditional preparation process.
Example 3 oxytocin [ -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: practice ofEXAMPLE 1 self-contained preparative column Load&Lock4002 50×250mm，
ODS-AQ 10μm 10nm
Oxytocin [ NH ]2]The crude product of impurities has a structural formula of
Reduced oxytocin [ NH ]2]Reduced oxytocin [ NH ] in impurity crude product solution2]The concentration of the crude product of the impurity is 1.5mg/ml, oxytocin [ -NH ]2]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
Mobile phase a was 0.02% acetic acid/water by volume, mobile phase B was 0.02% acetic acid/acetonitrile by volume, sample C1 was oxytocin [ -NH2]Crude solution of impurities, oxytocin [ -NH ] determined according to HPLC method2]The crude solution of the impurity had an HPLC purity of 73.13% and a mobile phase C2 of 20mM NH4Ac-NH4OH pH8.5 aqueous solution.
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 was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
Collecting the eluent with retention time of 80-89 min to obtain oxytocin [ -NH ]2]An impurity solution. oxytocin-NH measured according to HPLC method2]Purity of impurity HPLC is 99.12%, oxytocin [ -NH ]2]The removal rate of impurities in the crude impurity solution is 25.99 percent; collecting the eluent with retention time of 83-86 min to obtain oxytocin-NH2]The purity of the impurity HPLC was 99.78%.
Example 4 oxytocin [ -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: EXAMPLE 1 self-contained preparative column Load&Lock4002 50×250mm，
ODS-AQ 10μm 10nm
Oxytocin [ NH ]2]The structural formula of the impurity is
Reduced oxytocin [ NH ]2]Reduced oxytocin [ NH ] in impurity crude product solution2]The concentration of the crude product of the impurity is 0.8mg/ml, and oxytocin [ -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% acetic acid/water by volume, mobile phase B was 0.05% acetic acid/acetonitrile by volume, sample C1 was oxytocin [ -NH2]Crude solution of impurities, oxytocin [ -NH ] determined according to HPLC method2]The crude solution of the impurity had an HPLC purity of 76.36% and a mobile phase C2 of 20mM NH4Ac-NH4OH pH7.5 aqueous solution.
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 was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
Collecting the eluent with retention time of 80-89 min to obtain oxytocin [ -NH ]2]An impurity solution. oxytocin-NH measured according to HPLC method2]Purity of impurity HPLC is 99.22%, oxytocin [ -NH ]2]The impurity removal rate of the impurity crude product solution is 22.86 percent; collecting the eluent with retention time of 83-86 min to obtain oxytocin-NH2]The purity of the impurity HPLC was 99.85%.
Example 5 oxytocin [ -NH ]2]Mass spectrometric detection of impurities
Example measurement Using Waters micromass ZQ Single quadrupole electrospray Mass Spectrometry (ESI-MS)2. 3 and 4 to obtain oxytocin < - > -NH2]Impurities, the test conditions were: performing mass spectrometry by adopting an electrospray ionization (ESI) source in a positive ionization mode, wherein the ionization voltage of a capillary tube is 3.0kV, and the sampling taper hole voltage is 35 kV; the ion source temperature is 115 ℃, the desolventizing temperature is 350 ℃, the desolventizing nitrogen flow rate is 700L/h, the cone hole back flushing nitrogen flow rate is 50L/h, and the sweep range of the four-level rod is 50.0-1500 m/z.
The detection result is as follows: molecular ion Peak [ M + H]+Mass to charge ratio (M/z) of 1008.45, main ion fragment peak [ M +2H]2+The mass-to-charge ratio (m/z) is 504.72, and the mass-to-charge ratios are all in accordance with the theoretical value, namely oxytocin [ -NH ]2]The molecular weight of the impurity is 1008.20.
Claims (11)
1. Oxytocin [ -NH2]A method for purifying impurities, characterized by comprising the steps of: adopts high performance liquid phase reverse phase chromatography to convert oxytocin to NH2]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 super water-resistant filler is UniSil
ODS-AQ super water-resistant filler;
the reverse phase enrichment, reverse phase salt conversion and reverse phase purification are all completed in one-step reverse phase elution process, and the conditions of the reverse phase enrichment, the reverse phase salt conversion and the reverse phase purification are as follows:
wherein, 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 solution with the volume percentage of 0.005-0.1 percent, and the sample C1 is oxytocin [ -NH2]Crude impurity solution with a mobile phase C2 of 5-50 mM NH4Ac-NH4The pH value of the mobile phase C2 is 7.0-9.0, and the flow rate is 80-100 ml/min;
collecting the eluent with retention time of 80-89 min to obtain oxytocin [ -NH ]2]An impurity solution;
the oxytocin [ -NH ]2]Oxytocin < - > NH in crude impurity solution2]The structural formula of the impurity is
。
2. Oxytocin [ -NH ] as claimed in claim 12]A method for purifying impurities, characterized by comprising: the oxytocin [ -NH ]2]The crude solution of impurity is reduced oxytocin [ -NH synthesized by solid phase2]The crude product of the impurity is obtained by dissolution, dilution and oxidation.
3. Oxytocin [ -NH ] as claimed in claim 22]A method for purifying impurities, characterized by comprising: the reduction type oxytocin [ -NH ]2]Dissolving and diluting the crude impurity product to obtain reduced oxytocin < -NH2]Crude solution of impurities, the reduced oxytocin [ -NH ]2]Reduced oxytocin [ NH ] in impurity crude product solution2]The concentration of the crude product of the impurities is 0.1-4 mg/ml; dissolving the reduced oxytocin [ -NH ]2]The solvent for the crude impurity was 50% by volume acetic acid/water solution.
4. Oxytocin [ -NH ] according to claim 32]A method for purifying impurities, characterized by comprising: the reduction type oxytocin [ -NH ]2]Reduced oxytocin [ NH ] in impurity crude product solution2]The concentration of the crude product of the impurities is 0.5-2 mg/ml.
5. Oxytocin [ -NH ] as claimed in claim 12]A method for purifying impurities, characterized by comprising: the oxytocin [ -NH ]2]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
6. Oxytocin [ -NH ] as claimed in claim 12]Method for purifying impuritiesThe method is characterized in that: the aperture of the super water-resistant filler is 7-10 nm, and the particle size is 10 microns.
7. Oxytocin [ -NH ] as claimed in claim 12]A method for purifying impurities, characterized by comprising: the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
8. Oxytocin [ -NH ] as claimed in 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 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, said oxytocin [ -NH ]2]The HPLC purity of the crude impurity solution is 60-85%;
and/or collecting the eluent with the retention time of 83-86 min to obtain oxytocin [ -NH ]2]An impurity solution.
9. Oxytocin [ -NH ] as claimed in claim 82]A method for purifying impurities, characterized by comprising: the oxytocin [ -NH ]2]The HPLC purity of the crude impurity solution is 70-85%.
10. Oxytocin [ -NH ] as claimed in claim 82]A method for purifying impurities, characterized by comprising: the oxytocin [ -NH ]2]The HPLC purity of the crude impurity solution is 70-80%.
11. Oxytocin [ -NH ] as claimed in claim 12]A method for purifying impurities, characterized by comprising: completely replacing the eluent with a mobile phase C2 from a sample C1 in a period of 45-46 min; and completely replacing the eluent from the mobile phase C2 to the mobile phase A within 51-52 min.
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| CN106518977B (en) * | 2017-01-03 | 2019-12-31 | 上海上药第一生化药业有限公司 | Preparation method of oxytocin [5-Asp ] |
| US20190135859A1 (en) * | 2017-08-11 | 2019-05-09 | Ferring B.V. | Method of manufacturing a pharmaceutical composition |
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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 |
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