CN106478780B - Preparation method of oxytocin [4-Glu ] - Google Patents

Preparation method of oxytocin [4-Glu ] Download PDF

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CN106478780B
CN106478780B CN201710002006.6A CN201710002006A CN106478780B CN 106478780 B CN106478780 B CN 106478780B CN 201710002006 A CN201710002006 A CN 201710002006A CN 106478780 B CN106478780 B CN 106478780B
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oxytocin
glu
reverse phase
crude product
precursor
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CN106478780A (en
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江锡铭
丁金国
黄臻辉
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Add Medicine To First Biochemical Pharmaceutcal Corp Ltd In Shanghai
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Add Medicine To First Biochemical Pharmaceutcal Corp Ltd In Shanghai
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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

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Abstract

The invention discloses a preparation method of oxytocin [4-Glu ]. The preparation method comprises the following steps: performing reverse phase cyclization, reverse phase purification and reverse phase desalting on the oxytocin [4-Glu ] 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 [4-Glu ] precursor crude product is an oxytocin [4-Glu ] precursor crude product containing two free sulfydryl groups. The invention innovatively applies the cyclization, purification and desalination by a reversed-phase adsorption method, solves the problems of cyclization, purification and desalination at one time, optimizes the production process and is suitable for industrial continuous production.

Description

Preparation method of oxytocin [4-Glu ]
Technical Field
The invention relates to the field of biological pharmacy. More specifically, the invention relates to a preparation method of oxytocin [4-Glu ].
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).
Because the properties of some impurities in the synthesized polypeptide are very close to those of a target product, establishing a proper method to fully detect the impurities is a great difficulty in researching substances related to the synthesized polypeptide drugs.
Oxytocin [4-Glu ] in China at present]Most of the methods adopt solid-phase synthesis to protect oxytocin resin, and then obtain oxytocin [4-Glu ] through cracking and drying]Precursor crude product (Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2Trifluoroacetate salt), highly diluted cyclization, purification, salt conversion and other steps to finally obtain oxytocin [4-Glu ]]. The high dilution cyclization is very unfavorable for the later purification due to the dilute concentration and large volume of the sample. An efficient method for preparing disulfide bond-containing polypeptide drug substance analogs is still lacking, and therefore, development of new methods for preparing disulfide bond-containing polypeptides is still urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of oxytocin [4-Glu ] in order to overcome the defects of complex preparation process, low yield, dilute sample concentration and large volume of oxytocin [4-Glu ] in the prior art. The preparation method of oxytocin [4-Glu ] takes a polypeptide crude product containing a pair of free sulfydryl (-SH), namely an oxytocin [4-Glu ] precursor crude product, as an initial raw material, and prepares the oxytocin [4-Glu ] with high purity through the steps of high pressure liquid phase reverse phase chromatography cyclization, purification and desalination. The polypeptide of the invention is 1 key impurity in the preparation process of oxytocin, so that the polypeptide can be used as a standard reference substance in the detection process of oxytocin to perform qualitative and quantitative analysis on oxytocin and impurities, and has important significance for improving the quality standard of oxytocin and controlling the product quality.
The invention solves the technical problems through the following technical scheme:
the invention provides a preparation method of oxytocin [4-Glu ], which comprises the following steps: performing reverse phase cyclization, reverse phase purification and reverse phase desalting on the oxytocin [4-Glu ] 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 [4-Glu ] precursor crude product is an oxytocin [4-Glu ] precursor crude product containing two free sulfydryl groups;
the oxytocin [4-Glu]Is composed of
Wherein, the oxytocin [4-Glu ] is]The precursor crude product is preferably oxytocin [4-Glu prepared by cracking and drying by adopting a solid phase synthesis method]A precursor crude product with HPLC purity of 60-90%; the oxytocin [4-Glu]The structural formula of the precursor crude product is Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2The trifluoroacetic acid salt of (1).
The oxytocin [4-Glu ] precursor crude product solution is preferably 5g/L of oxytocin [4-Glu ] precursor crude product dissolved in 5% by volume of acetonitrile water solution.
In the invention, the reverse phase cyclization, the reverse phase purification and the reverse phase desalination are all completed in a one-step reverse phase elution process.
Wherein, the conditions of reverse phase cyclization, reverse phase purification and reverse phase desalting are preferably as follows: the mobile phase A1 is pure water, A2 is H with a volume percentage of 0.01-0.05% (preferably 0.02-0.03%)2O2NaOH aqueous solution with pH of 7.5-9.0, wherein the mobile phase B is acetonitrile, and the mobile phase C is oxytocin [4-Glu]The flow rate of the precursor crude product solution is 80-110 mL/min (preferably 100mL/min), and the detection wavelength is 220 nm;
carrying out on-line sample loading and elution according to the conditions of the following table, wherein the percentage is volume percentage;
step of elution Elution time Eluent
1 0～10min 100％C
2 10～25min 95％A1+5％B
3 25.1～30min 95％A2+5％B
4 30.1～35 95％A1+5％B
5 35～50min 95％A1+5％B→80％A1+20％B
6 50～65min 80％A1+20％B→73％A1+27％B
7 65～75min 50％A1+50％B
8 75～80min 95％A1+5％B
Collecting the eluent with the retention time of 50-65 min to obtain the oxytocin [4-Glu ] solution.
Wherein, the packing conditions of the high performance liquid reverse phase chromatography are preferably as follows: the filler is Kromasil silica gel C18, the pore diameter is 10nm, and the particle size is 10 μm.
Oxytocin [4-Glu ] is a polypeptide substance which is unstable and easy to degrade under alkaline conditions, and particularly under a strong alkaline environment, the concentration and time of alkaline elution are comprehensively considered, so that the damage and loss of a sample in the desalting process are reduced.
The innovation point of the invention is that the polypeptide pure product is obtained by one-step phase reversal of cyclization, purification and desalination, the oxytocin [4-Glu ] precursor crude product contains two free sulfydryl groups, the traditional process is performed by cyclization and purification step by step, the cyclization volume is large, the purification difficulty is increased, and the latest application of the silica gel C18 filler is designed for the rapid and efficient cyclization and preparation process. The invention innovatively applies the reverse phase adsorption method for cyclization, purification and desalination, and solves the problems of cyclization, purification and desalination at one time.
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 cyclization method, firstly, a reduced polypeptide crude product is adsorbed on a stationary phase, weakly-bound acid radical ions are eluted on a chromatographic column in a neutral manner by utilizing the hydrophobic binding of the polypeptide and a reverse phase filler, and H-containing H with pH bias alkali is adopted2O2The two sulfydryl groups are promoted to form disulfide bonds by washing the mobile phase to obtain a crude target polypeptide product, and the sample is retained on a chromatographic column.
(2) The method adopts on-line cyclization, the sample subjected to cyclization avoids elution, and gradient elution purification can be carried out after mobile phase conversion directly to obtain a final pure product, so that the method is suitable for continuous production.
(3) The invention creatively uses the one-step method of reversed phase adsorption cyclization, purification and desalination to prepare the pure polypeptide product, optimizes the production process and is suitable for industrial continuous production.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following examples oxytocin [4-Glu]Is composed of
Example 1(HPLC method for detecting purity and quantitation of oxytocin [4-Glu ] precursor crude product and purified intermediate solution)
The instrument comprises the following steps: agilent 1200 high performance liquid chromatograph
Separating the column: waters Xbridge-C18, 4.6X 150 mm, 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 1.0 mL/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 28.0～28.1min 100％A→35％A+65％B
5 28.1～32min 35％A+65％B
Example 2(50mm ID L & L4002 column packing)
Application of Load&The Lock dynamic axial compression and static locking technology is characterized in that a filler is silica gel C18(Kromasil C18), the pore diameter is 10nm, the particle size is 10 mu m, the packing density is 0.66 g/mL, the column is filled until the pressure of a column bed is 133bar, a Varian chromatography filling system is adopted, 325g of dry powder filler and 650mL of isopropanol are stirred and homogenized, and then the mixture is poured into L with the inner diameter of 50mm&L4002 column preparation, compression ratio 1.5:1, carrier gas N2The carrier gas pressure was adjusted to 200bar gauge pressure and dynamic axial compression to 25.5cm height of the bed was used as a preparative column for the reverse phase cyclization, reverse phase purification and reverse phase desalination protocol.
EXAMPLE 3 reverse phase cyclization, reverse phase purification and reverse phase desalting of crude oxytocin [4-Glu ] precursor starting Material
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: example 2 self-contained preparative column Load & Lock 400250X 255mm, C1810 μm10nm
Oxytocin [4-Glu]The precursor crude product is oxytocin [4-Glu prepared by cracking and drying by adopting a solid-phase synthesis method]The precursor crude product has a structural formula of Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2The trifluoroacetic acid salt of (1).
The oxytocin [4-Glu ] precursor crude product solution is 5g/L solution formed by dissolving the oxytocin [4-Glu ] precursor crude product in 5% by volume of acetonitrile water solution.
The mobile phase A1 is purified water, A2 is 0.02 volume percent of H2O2NaOH aqueous solution with pH of 7.5, mobile phase B is acetonitrile, and mobile phase C is oxytocin [4-Glu]The crude precursor solution had an HPLC purity of 78.50% and a retention time of 9.46min, as determined in example 1.
The reverse phase cyclization, reverse phase purification and reverse phase desalting 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.
Step of elution Elution time Eluent
1 0～10min 100％C
2 10～25min 95％A1+5％B
3 25.1～30min 95％A2+5％B
4 30.1～35 95％A1+5％B
5 35～50min 95％A1+5％B→80％A1+20％B
6 50～65min 80％A1+20％B→73％A1+27％B
7 65～75min 50％A1+50％B
8 75～80min 95％A1+5％B
And collecting the eluent with the retention time of 50-65 min to obtain oxytocin [4-Glu ] solution, and determining that the HPLC purity is 99.49% and the retention time is 6.20min according to the method of the embodiment 1.
EXAMPLE 4 reverse phase cyclization, reverse phase purification and reverse phase desalting of crude oxytocin [4-Glu ] precursor starting Material
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: example 2 self-contained preparative column Load & Lock 400250X 255mm, C1810 μm10nm
Oxytocin [4-Glu]The precursor crude product is oxytocin [4-Glu prepared by cracking and drying by adopting a solid-phase synthesis method]The precursor crude product has a structural formula of Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2The trifluoroacetic acid salt of (1).
The oxytocin [4-Glu ] precursor crude product solution is 5g/L solution formed by dissolving the oxytocin [4-Glu ] precursor crude product in 5% by volume of acetonitrile water solution.
The mobile phase A1 is purified water, A2 is 0.03 percent by volume of H2O2NaOH aqueous solution with pH of 9.0, mobile phase B is acetonitrile, and mobile phase C is oxytocin [4-Glu]The crude precursor solution had an HPLC purity of 78.50% and a retention time of 9.46min, as determined in example 1.
The reverse phase cyclization, reverse phase purification and reverse phase desalting 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.
Step of elution Elution time Eluent
1 0～10min 100％C
2 10～25min 95％A1+5％B
3 25.1～30min 95％A2+5％B
4 30.1～35 95％A1+5％B
5 35～50min 95％A1+5％B→80％A1+20％B
6 50～65min 80％A1+20％B→73％A1+27％B
7 65～75min 50％A1+50％B
8 75～80min 95％A1+5％B
And collecting the eluent with the retention time of 50-65 min to obtain oxytocin [4-Glu ] solution, and determining that the HPLC purity is 99.57% and the retention time is 6.27min according to the method of the embodiment 1.
Example 5 Mass spectrometric detection of oxytocin [4-Glu ]
Oxytocin [4-Glu ] obtained in examples 3 and 4 was measured by waters micromass ZQ single quadrupole electrospray mass spectrometry (ESI-MS)]Molecular mass peak of (1) [ M +1 ]]+Found 1009.28, main ion fragment Peak [ M +2 ]]2+The measured values 505.17 all met the theoretical value 1008.2.

Claims (8)

1. A preparation method of oxytocin [4-Glu ] comprises the following steps: performing reverse phase cyclization, reverse phase purification and reverse phase desalting on the oxytocin [4-Glu ] 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 [4-Glu ] precursor crude product is an oxytocin [4-Glu ] precursor crude product containing two free sulfydryl groups;
the oxytocin [4-Glu]Is composed of
The conditions of reverse phase cyclization, reverse phase purification and reverse phase desalting are as follows: the mobile phase A1 is pure water, A2 is H with the volume percentage of 0.01-0.05%2O2The pH of the NaOH aqueous solution is 7.5-9.0, the mobile phase B is acetonitrile, and the mobile phase C is oxytocin [4-Glu ]]The flow rate of the precursor crude product solution is 80-110 mL/min, and the detection wavelength is 220 nm;
carrying out on-line sample loading and elution according to the conditions of the following table, wherein the percentage is volume percentage;
step of elution Elution time Eluent 1 0～10min 100％C 2 10～25min 95％A1+5％B 3 25.1～30min 95％A2+5％B 4 30.1～35 95％A1+5％B 5 35～50min 95％A1+5％B→80％A1+20％B 6 50～65min 80％A1+20％B→73％A1+27％B 7 65～75min 50％A1+50％B 8 75～80min 95％A1+5％B
Collecting the eluent with the retention time of 50-65 min to obtain the oxytocin [4-Glu ] solution.
2. The preparation method of claim 1, wherein the oxytocin [4-Glu ] precursor crude product is prepared by cracking and drying through a solid-phase synthesis method, and the HPLC purity is 60-90%.
3. The method according to claim 1, wherein said oxytocin [4-Glu ] is]The structural formula of the precursor crude product is Cys-Tyr-Ile-Glu-Asn-Cys-Pro-Leu-Gly-NH2The trifluoroacetic acid salt of (1).
4. The method according to claim 1, wherein the crude oxytocin [4-Glu ] precursor solution is 5g/L of a solution of the crude oxytocin [4-Glu ] precursor dissolved in 5% by volume of acetonitrile water.
5. The method of claim 1, wherein the reverse phase cyclization, the reverse phase purification and the reverse phase desalting are all completed in a one-step reverse phase elution process.
6. The method according to claim 1, wherein A2 is H0.02-0.03 vol%2O2The pH of the NaOH aqueous solution is 7.5-9.0.
7. The method of claim 1, wherein the flow rate is 100 mL/min.
8. The method of claim 1, wherein the high performance liquid reverse phase chromatography is carried out under the following packing conditions: the filler is Kromasil silica gel C18, the pore diameter is 10nm, and the particle size is 10 μm.
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Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110016072B (en) * 2019-05-07 2022-03-15 上海上药第一生化药业有限公司 Method for refining oxytocin
CN110041405B (en) * 2019-05-07 2021-04-13 上海上药第一生化药业有限公司 Method for refining oxytocin [5-Asp ] impurity
CN110078797B (en) * 2019-05-07 2021-04-13 上海上药第一生化药业有限公司 Method for refining oxytocin [4-Glu ] impurity
CN110078796B (en) * 2019-05-07 2021-04-13 上海上药第一生化药业有限公司 Method for refining oxytocin [4-Glu,5-Asp ] impurity
CN110028556B (en) * 2019-05-07 2021-04-13 上海上药第一生化药业有限公司 Method for refining oxytocin impurity [ -NH2]

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CN101235081A (en) * 2008-03-10 2008-08-06 无锡市凯利药业有限公司 Method for preparing oxytocin
CN103374054A (en) * 2012-04-28 2013-10-30 上海第一生化药业有限公司 One-step method based solid-phase polypeptide synthesis method
CN103980351A (en) * 2014-05-27 2014-08-13 上海第一生化药业有限公司 Method for preparing vasopressin and vasopressin tannate
CN104672308A (en) * 2014-12-23 2015-06-03 青岛康原药业有限公司 Method for preparing vasopressin tannate

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CN101235081A (en) * 2008-03-10 2008-08-06 无锡市凯利药业有限公司 Method for preparing oxytocin
CN103374054A (en) * 2012-04-28 2013-10-30 上海第一生化药业有限公司 One-step method based solid-phase polypeptide synthesis method
CN103980351A (en) * 2014-05-27 2014-08-13 上海第一生化药业有限公司 Method for preparing vasopressin and vasopressin tannate
CN104672308A (en) * 2014-12-23 2015-06-03 青岛康原药业有限公司 Method for preparing vasopressin tannate

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