CN110016072B - Method for refining oxytocin - Google Patents
Method for refining oxytocin Download PDFInfo
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- CN110016072B CN110016072B CN201910375969.XA CN201910375969A CN110016072B CN 110016072 B CN110016072 B CN 110016072B CN 201910375969 A CN201910375969 A CN 201910375969A CN 110016072 B CN110016072 B CN 110016072B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
Abstract
The invention discloses a method for refining oxytocin, which comprises the following steps: sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the oxytocin crude product solution by adopting a high performance liquid reversed-phase chromatography; the oxytocin crude product solution is obtained by oxidizing a reduction-type oxytocin crude product solution synthesized by a solid phase; the filler of the high performance liquid phase reverse phase chromatography is super water-resistant filler. The invention adopts one-step method of on-line reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification to prepare the pure polypeptide product, optimizes the production process, has the advantages that the mobile phase in the stages of column balance, sample loading enrichment and salt conversion is aqueous solution, is environment-friendly and pollution-free, and effluent waste liquid can be directly treated and recycled.
Description
Technical Field
The invention relates to a method for refining oxytocin.
Background
Oxytocin, also known as Oxytocin, is known as oxyytocin and has the structural formula:
the molecular formula is: c43H66N12O12S2Molecular weight of 1007.19
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.
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 CN106674332A discloses a preparation method of oxytocin. The preparation method comprises the following steps: performing reverse phase cyclization, reverse phase purification and reverse phase desalting on the oxytocin precursor crude product solution by adopting a high performance liquid reverse phase chromatography in sequence; the filler of the high performance liquid reverse phase chromatography is silica gel C18; the oxytocin precursor crude product is an oxytocin precursor crude product containing two free sulfydryl groups. In the preparation process, a reduced oxytocin precursor crude product is used as an initial raw material, a large amount of mobile phase containing an organic solvent is needed when 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 subsequent waste liquid is high.
Chinese patent document CN1990501A discloses a method for solid-phase synthesis of oxytocin, which takes Rink Amide or Rink MBHA resin as a starting material and TBTU/HOBt or HABU/HOBt as a condensing agent to gradually synthesize oxytocin; then cutting, adding air or hydrogen peroxide (10)-3～10-4mol/L) solution is oxidized, intermolecular reaction is easy to generate linear or cyclic dimerization and multimer, the method has the defects that oligomerization reaction among peptide molecules is easy to cause serious reduction of yield and purity of cyclic peptide, and mobile phases in the preparation process of the method are all organic solvents, so that the generated hazardous waste liquid is high in treatment cost and difficult to recycle.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of high treatment cost and low economy of waste liquid caused by large amount of hazardous waste liquid which is organic hazardous waste liquid in the prior art, and provides a method for refining oxytocin. Most of waste liquid generated in the process of purifying a target product by the method for refining oxytocin can be directly recycled by 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 method for refining oxytocin, which comprises the following steps:
sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the oxytocin 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 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 is0.005-0.1% by volume of acetic acid/water, 0.005-0.1% by volume of acetic acid/acetonitrile as mobile phase B, 5-50 mM NH 2 as sample C1 and crude oxytocin solution as sample C4Ac-NH4The pH value of the mobile phase C2 is 7.0-9.0, and the flow rate is 80-100 ml/min;
and collecting the eluent with the retention time of 95-105 min to obtain the oxytocin solution.
In the invention, the oxytocin crude product solution is prepared by dissolving, diluting and oxidizing a reduction oxytocin crude product synthesized by a solid phase.
The preparation method of the oxytocin 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 reduced oxytocin crude product; dissolving the reduction type oxytocin crude product with 50% acetic acid/water, and diluting with water to obtain reduction type oxytocin crude product solution; and (2) adjusting the pH value of the reduction type oxytocin crude product solution to 7.0-9.0 by using an alkaline substance, adding 30% hydrogen peroxide for oxidation, wherein 0.5ml of 30% hydrogen peroxide is added into each gram of reduction type oxytocin crude product to obtain an oxidation type oxytocin crude product solution, namely the oxytocin crude product solution.
Wherein 50% acetic acid/water can fully dissolve the crude reduced oxytocin product.
Wherein the concentration of the reduced oxytocin crude product 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 invention, the structural formula of oxytocin in the oxytocin crude product solution is shown as
The solvent in the oxytocin crude product solution is trifluoro-containingAcetic acid and aqueous acetic acid solutions.
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 pore diameter of 10nm and particle diameter of 10 μm, loading to column bed pressure of 1000psi, adopting Varian chromatography loading 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 the HPLC purity of the oxytocin crude product solution is 60-85%, preferably 70-85%, more preferably 70-80%;
and/or collecting the eluent with the retention time of 98-101 min to obtain the oxytocin solution.
In the invention, the eluent is completely changed from a sample C1 to a mobile phase C2 within 50-51 min; and completely replacing the eluent from the mobile phase C2 to the mobile phase A during 71-72 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 115-116 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 116-125 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 removing trifluoroacetate in a crude oxytocin product, step (3) is a process for removing ammonium ions in step (2), and the reversed-phase purification is steps (4) and (5), wherein step (4) is a process for removing weak adsorbed impurities.
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 reduced 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 is a polypeptide substance which is unstable and easy to degrade under the condition of high pH, and particularly under the alkaline environment, the pH and the time of salt transfer elution are comprehensively considered, so that the damage and the loss of a sample in the salt transfer process are reduced.
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.
(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.
Detecting the purity of the oxytocin crude product and the purified product solution by an HPLC method:
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, and B is 0.02M KH2PO4pH3.0 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, the crude oxytocin solution is a reduced oxytocin crude product synthesized by solid phase through dissolution and dilutionIs obtained by releasing and oxidizing. The solid phase synthesis method comprises the following steps: taking Rink Amide MBHA resin as an initial raw material, taking amino acid protected by Fmoc as a monomer, taking HOBt/DIC as a condensing agent, and sequentially connecting the amino acid one by one; adding a peptide cutting reagent for peptide cutting, adding methyl tert-butyl ether for precipitation, and obtaining a crude product of the reduction type oxytocin. 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 adjust the pH of the reduction type oxytocin crude product solution to 7.0-9.0 by using alkaline substances, and add hydrogen peroxide with the volume percentage of 30% to carry out the oxidation process. The dosage of the hydrogen peroxide is 0.5mL/1g of the reduced oxytocin crude product. 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 reverse phase enrichment, reverse phase desalting and reverse phase purification of oxytocin crude 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&Lock400250×250mm，
ODS-AQ10μm10nm
The structural formula of oxytocin is
The concentration of the reduction type oxytocin crude product in the reduction type oxytocin crude product solution is 1mg/ml, and the solvent in the oxytocin crude product solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is 0.02 percent of acetic acid/water by volume, the mobile phase B is 0.02 percent of acetic acid/acetonitrile by volume, the sample C1 is a crude oxytocin solution, the HPLC purity of the crude oxytocin solution is 71.23 percent determined by an HPLC method, and the mobile phase C2 is 10mM 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.
And collecting the eluent with the retention time of 95-105 min to obtain the oxytocin solution. Purity of oxytocin HPLC determined according to HPLC method was 99.63%; and collecting the eluent with the retention time of 98-101 min to obtain the oxytocin HPLC with the purity of 99.85%.
The one-step method of the embodiment completes the salt conversion and purification of the oxytocin crude product solution, the eluent in the elution steps (1) to (3) is aqueous solution, the method is environment-friendly and pollution-free, the effluent liquid of the method is directly discharged to a sewage treatment station, and can be recycled after simple treatment, compared with the traditional preparation process, the method greatly reduces the generation amount of dangerous waste liquid, and saves the environment.
Example 3 reverse phase enrichment, reverse phase desalting and reverse phase purification of oxytocin crude 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&Lock400250×250mm，
ODS-AQ10μm10nm
The structural formula of oxytocin is
The concentration of the reduction type oxytocin crude product in the reduction type oxytocin crude product solution is 1.5mg/ml, and the solvent in the oxytocin crude product solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is 0.05 percent of acetic acid/water by volume, the mobile phase B is 0.05 percent of acetic acid/acetonitrile by volume, the sample C1 is a oxytocin crude product solution, the HPLC purity of the oxytocin crude product solution is 71.03 percent according to the HPLC method, and the mobile phase C2 is 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.
And collecting the eluent with the retention time of 95-105 min to obtain the oxytocin solution. The purity of oxytocin by HPLC method was 99.43%; and collecting the eluent with the retention time of 98-101 min to obtain the oxytocin HPLC with the purity of 99.82%.
Example 4 reverse phase enrichment, reverse phase desalting and reverse phase purification of oxytocin crude 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&Lock400250×250mm，
ODS-AQ10μm10nm
The structural formula of oxytocin is
The concentration of the reduction type oxytocin crude product in the reduction type oxytocin crude product solution is 0.8mg/ml, and the solvent in the oxytocin crude product solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is 0.05 percent of acetic acid/water by volume, the mobile phase B is 0.05 percent of acetic acid/acetonitrile by volume, the sample C1 is a oxytocin crude product solution, the HPLC purity of the oxytocin crude product solution is 70.46 percent according to the HPLC method, and the mobile phase C2 is 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.
And collecting the eluent with the retention time of 95-105 min to obtain the oxytocin solution. The HPLC purity of oxytocin determined according to the HPLC method was 99.54%; and collecting the eluent with the retention time of 98-101 min to obtain the oxytocin with the HPLC purity of 99.87%.
Example 5 Mass Spectrometry detection of oxytocin
The oxytocin 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 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 1007.45, main ion fragment peak [ M +2H]2+The mass to charge ratio (m/z) was 504.22, which all fit the theoretical value, i.e. the molecular weight of oxytocin was 1007.19.
Claims (11)
1. A method for refining oxytocin is characterized by comprising the following steps: sequentially carrying out reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification on the oxytocin crude product solution by adopting a high performance liquid reversed-phase chromatography;
the filler of the high performance liquid reverse phase chromatography is
ODS-AQ super water-resistant filler; the structural formula of oxytocin in the oxytocin crude product solution is shown as
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 volume percentage of 0.005-0.1%, the mobile phase B is acetic acid/acetonitrile solution with volume percentage of 0.005-0.1%, the sample C1 is the oxytocin 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 is 80-100 ml/min;
and collecting the eluent with the retention time of 95-105 min to obtain the oxytocin solution.
2. A method for refining oxytocin according to claim 1, characterized in that: the oxytocin crude product solution is obtained by dissolving, diluting and oxidizing a reduction oxytocin crude product synthesized by a solid phase.
3. A method for refining oxytocin according to claim 2, characterized in that: dissolving and diluting the reduction type oxytocin crude product to obtain a reduction type oxytocin crude product solution, wherein the concentration of the reduction type oxytocin crude product in the reduction type oxytocin crude product solution is 0.1-4 mg/ml; the solvent for dissolving the crude reduced oxytocin is acetic acid/water solution with the volume percentage of 50%.
4. The refining method of oxytocin according to claim 3, characterized in that the concentration of the crude reduced oxytocin in the crude reduced oxytocin solution is 0.5-2 mg/ml.
5. A method for refining oxytocin according to claim 1, characterized in that: the solvent in the oxytocin crude product solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
6. A method for refining oxytocin according to claim 1, characterized in that: the aperture of the super water-resistant filler is 7-10 nm, and the particle size is 10 microns.
7. A method for refining oxytocin according to claim 1, characterized in that: the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
8. A method for refining oxytocin according to claim 1, characterized in that: the mobile phase A is an acetic acid/water solution with the volume percentage of 0.02-0.05%;
and/or the mobile phase B is an acetic acid/acetonitrile solution with the volume percentage of 0.02-0.05%;
and/or the mobile phase C2 is 10-20 mM NH4Ac-NH4An aqueous OH solution;
and/or the pH of the mobile phase C2 is 7.5-8.5;
and/or the HPLC purity of the oxytocin crude product solution is 60-85%;
and/or collecting the eluent with the retention time of 98-101 min to obtain the oxytocin solution.
9. The refining method of oxytocin according to claim 8, characterized in that the HPLC purity of the crude oxytocin solution is 70% -85%.
10. The refining method of oxytocin according to claim 8, characterized in that the HPLC purity of the crude oxytocin solution is 70% -80%.
11. A method for refining oxytocin according to claim 1, characterized in that: during 50-51 min, the eluent C1 was completely changed to C2, and during 71-72 min, the eluent C2 was completely changed to A.
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| CN113827700A (en) * | 2021-09-30 | 2021-12-24 | 上海上药第一生化药业有限公司 | Oxytocin-containing pharmaceutical composition, lyophilized powder thereof and oxytocin purification method |
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