CN106674332A - Preparation method of oxytocin - Google Patents
Preparation method of oxytocin Download PDFInfo
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- CN106674332A CN106674332A CN201710001528.4A CN201710001528A CN106674332A CN 106674332 A CN106674332 A CN 106674332A CN 201710001528 A CN201710001528 A CN 201710001528A CN 106674332 A CN106674332 A CN 106674332A
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- oxytocin
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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. The preparation method comprises steps as follows: an oxytocin precursor crude product solution is sequentially subjected to reversed-phase cyclization, reversed-phase purification and reversed-phase desalination with high-performance reversed-phase liquid chromatography; filler used in the high-performance reversed-phase liquid chromatography method is silica gel C18; an oxytocin precursor crude product contains two free sulfydryl groups. The reversed-phase adsorbing cyclization, purification and desalination one-step method is applied in the preparation method for preparation of a polypeptide pure product, the production process is optimized, and the preparation method is suitable for industrial continuous production.
Description
Technical field
The present invention relates to field of biological pharmacy.More particularly it relates to a kind of preparation method of oxytocin.
Background technology
Oxytocin, also known as oxytocin, English entitled Oxytocin, structural formula：
Molecular formula is：C43H66N12O12S2, molecular weight is 1007.2
Oxytocin be used for induced labor, hasten parturition, the metrorrhagia that puerperal and post-abortion cause because uterine atony or contracting abdomen are bad；
Understand Placenta Hominiss reserve function (oxytocin enrages test)；Collunarium can promote milk ejection.Oxytocin energy indirect stimulation uterine smooth muscle is received
Contracting, simulates the effect of eutocous uterine contraction, causes cervical dilatation, uterus to the reaction of oxytocin in During Pregnancy by
It is cumulative to add, peak is reached when mature.Oxytocin can also pierce mammotropic smooth muscle contraction, contribute to milk and discharge from breast, but and
The galactopoiesiss amount of mammary gland is not increased.
At present country's oxytocin using solid phase synthesis protected oxytocin resin before this mostly, then obtained contracting palace through cracking is dry
Plain precursor crude product (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2Trifluoroacetate), high dilution cyclisation,
Purification, turns the steps such as salt, finally gives oxytocin.High dilution cyclisation is dilute due to sample concentration, bulky, pure for the later stage
It is very unfavorable to change.Also lack now it is a kind of it is effective prepare containing disulfide bond polypeptide crude drug method, therefore still in the urgent need to
The new preparation method containing disulfide bond polypeptide of exploitation.
The content of the invention
The technical problem to be solved be in order to overcome prior art in oxytocin complicated process of preparation, receive
Rate is low, sample concentration is dilute, bulky defect, and provides a kind of preparation method of oxytocin.The oxytocin of the present invention
Preparation method is with the polypeptide crude product of a pair free two sulfydryls (- SH) --- and oxytocin precursor crude product passes through efficient as initiation material
The cyclisation of liquid phase reversed phase chromatography, purification and salt-removal steps, the method for preparing highly purified oxytocin.
The present invention is to solve above-mentioned technical problem by the following technical programs：
The invention provides a kind of preparation method of oxytocin, it comprises the steps：Using efficient liquid phase reversed phase chromatography
Oxytocin precursor crude product solution is carried out successively anti-phase cyclisation, anti-phase purification, anti-phase desalination by method, you can；The anti-phase color of efficient liquid phase
The filler of spectrometry is silica gel C18；
Described oxytocin precursor crude product is the oxytocin precursor crude product containing two free sulfhydryl groups.
Wherein, described oxytocin precursor crude product is preferably dried obtained contracting palace using solid-phase synthesis through cracking
Plain precursor crude product, HPLC purity is 60~90%；The structural formula of described oxytocin precursor crude product is Cys-Tyr-Ile-Gln-
Asn-Cys-Pro-Leu-Gly-NH2Trifluoroacetate.
Wherein, described oxytocin precursor crude product solution is preferably oxytocin precursor crude product and is dissolved in concentration of volume percent
For the 5g/L solution of 5% acetonitrile solution formation.
In the present invention, described anti-phase cyclisation, anti-phase purification, anti-phase desalination are complete in the anti-phase elution process of a step
Into.
Wherein, described anti-phase cyclisation, anti-phase purification, the condition of anti-phase desalination are preferably as follows：Mobile phase A 1 be pure water, A2
For the H of percent by volume 0.01~0.05% (preferably 0.02~0.03%)2O2PH is 7.5~9.0 NaOH aqueous solutions,
Mobile phase B is acetonitrile, and mobile phase C is described oxytocin precursor crude product solution, flow velocity be 80~110mL/min (preferably
100mL/min), Detection wavelength is 220nm；
Condition according to following table carries out online loading, eluting, and percentage ratio is percent by volume；
Collect the eluent that retention time is 50~65min and obtain oxytocin solution.
Wherein, the filling condition of described efficient liquid phase reverse-phase chromatography is preferably as follows：Filler is Kromasil silica gel
C18, aperture 10nm, 10 μm of particle diameter.
Oxytocin is a kind of peptide material, unstable in the basic conditions, degradable, especially under strong alkali environment, this
The de- concentration of bright integrated survey alkali cleaning and time, to ensure to reduce the destruction and loss of sample in desalination processes.
Innovative point of the present invention is will cyclisation, purification and the step of desalination one is anti-phase obtains polypeptide sterling, oxytocin precursor crude product
In contain two free sulfhydryl groups, traditional handicraft cyclisation and purification substep carry out, and be cyclized it is bulky, increased the difficulty of purification
Degree, for cyclisation rapidly and efficiently and preparation technology, designs the more recent application of silica gel C18 fillers.Novelty of the present invention is used
The cyclisation of reverse phase absorption method, purification and desalination, disposably solve the problems, such as to be cyclized, purification and turn salt.
On the basis of common sense in the field is met, above-mentioned each optimum condition, can combination in any, obtain final product each preferable reality of the present invention
Example.
Agents useful for same of the present invention and raw material are commercially available.
The present invention positive effect be：
(1) present invention is first adsorbed onto reduced form polypeptide crude product in fixing phase, using polypeptide using the method for online cyclisation
With the hydrophobic binding of reverse phase filler, the acid ion of neutral eluting weak binding on a column, and using pH meta-alkalis containing H2O2's
Mobile phase is rinsed, and promotes two sulfydryls into disulfide bond, obtains target polypeptides crude product, and sample retains on a column.
(2) using online cyclisation, the sample of cyclisation avoids eluting to the present invention, and can directly convert carries out gradient and wash after mobile phase
De- purification, obtains final sterling, is adapted to continuous production.
(3) present invention has innovatively used reverse phase absorption cyclisation, purification, desalination one-step method that polypeptide sterling, optimization is obtained
Production technology, suitable industrialization is continuously produced.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Among applying a scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product description is selected.
Embodiment 1 (HPLC methods detect oxytocin precursor crude product and purification midbody solution purity and quantitative)
Instrument：The high performance liquid chromatographs of Agilent 1200
Detached dowel：Waters XBridge-C18,4.6 × 150mm, 5 μm
Mobile phase：A is the acetonitrile solution of percent by volume 50%, and B is 0.02M KH2PO4The aqueous solutions of pH 3.0, flow velocity is
1.0mL/min, Detection wavelength is 220nm, room temperature detection, and gradient see the table below shown, and percentage ratio is percent by volume.
| Elution step | 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 |
Embodiment 2 (50mm internal diameter L＆L4002 prepare post filling)
With Load＆Lock dynamic axial compressions and static locking technology, filler is silica gel C18 (Kromasil C18),
Aperture 10nm, 10 μm of particle diameter fills column density 0.66g/mL, is filled to post bed pressure 133bar, is using the filling of Varian chromatographs
System, 325g dry powder fillers after the stirring homogenate of 650mL isopropanols, pour internal diameter 50mm L＆L4002 into and prepare post, and compression ratio is 1.5:
1, carrier gas is N2, adjust nebulizer gas pressure and cause oil pressure meter pressure for 200bar, dynamic axial compression to post bed height 25.5cm, work
Post is prepared used by anti-phase cyclisation, anti-phase purification and anti-phase desalination scheme.
The anti-phase cyclisation of the oxytocin precursor crude material of embodiment 3, anti-phase purification and anti-phase desalination
Instrument：Varian SD-1 high-pressure liquid phase preparation systems
Chromatographic column：The self-chambering of embodiment 2 prepares post 50 × 260mm of Load＆Lock4002,10 μm of 10nm of C18
Oxytocin precursor crude product is to be dried obtained oxytocin precursor crude product, structural formula through cracking using solid-phase synthesis
For Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2Trifluoroacetate.
Oxytocin precursor crude product solution is the acetonitrile water that above-mentioned oxytocin precursor crude product is dissolved in that concentration of volume percent is 5%
The 5g/L solution that solution is formed.
Mobile phase A 1 is purified water, and A2 is the H of percent by volume 0.03%2O2PH is 7.5 NaOH aqueous solutions, Mobile phase B
For acetonitrile, mobile phase C is described oxytocin precursor crude product solution, and its HPLC purity is determined as described in Example 1 is
71.50%, retention time is 9.61min.
The anti-phase cyclisation of the present embodiment, anti-phase purification and anti-phase desalination condition are as follows：Flow velocity 100mL/min, 220nm are examined
Survey, purification gradient see the table below shown, and percentage ratio is percent by volume.
| Elution step | 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 |
Collect the eluent that retention time is 50~65min and obtain oxytocin solution, it is determined as described in Example 1
HPLC purity is 99.66%, and retention time is 6.35min.
The anti-phase cyclisation of the oxytocin precursor crude material of embodiment 4, anti-phase purification and anti-phase desalination
Instrument：Varian SD-1 high-pressure liquid phase preparation systems
Chromatographic column：The self-chambering of embodiment 2 prepares post 50 × 255mm of Load＆Lock4002,10 μm of 10nm of C18
Oxytocin precursor crude product is to be dried obtained oxytocin precursor crude product, structural formula through cracking using solid-phase synthesis
For Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2Trifluoroacetate.
Oxytocin precursor crude product solution is the acetonitrile water that above-mentioned oxytocin precursor crude product is dissolved in that concentration of volume percent is 5%
The 5g/L solution that solution is formed.
Mobile phase A 1 is purified water, and A2 is the H of percent by volume 0.022O2PH is 9.0 NaOH aqueous solutions, and Mobile phase B is
Acetonitrile, mobile phase C is described oxytocin precursor crude product solution, and its HPLC purity is determined as described in Example 1 is
71.50%, retention time is 9.61min.
The anti-phase cyclisation of the present embodiment, anti-phase purification and anti-phase desalination condition are as follows：Flow velocity 100mL/min, 220nm are examined
Survey, purification gradient see the table below shown, and percentage ratio is percent by volume.
| Elution step | 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 |
Collect the eluent that retention time is 50~65min and obtain oxytocin solution, it is determined as described in Example 1
HPLC purity is 99.69%, and retention time is 6.37min.
Embodiment 5 (Mass Spectrometer Method of oxytocin)
Determine what enforcement 3,4 was obtained using waters micromass ZQ substances level Four bar Electrospray Mass Spectrometry (ESI-MS)
The molecular mass peak [M+1] of oxytocin+Measured value 1008.25, leading ion fragment peak [M+2]2+Measured value 504.68, all meets
Theoretical value 1007.2.
Claims (9)
1. a kind of preparation method of oxytocin, it comprises the steps：Using efficient liquid phase reverse-phase chromatography by oxytocin precursor
Crude product solution carries out successively anti-phase cyclisation, anti-phase purification, anti-phase desalination, you can；The filler of efficient liquid phase reverse-phase chromatography is silicon
Glue C18；
Described oxytocin precursor crude product is the oxytocin precursor crude product containing two free sulfhydryl groups.
2. preparation method as claimed in claim 1, it is characterised in that described oxytocin precursor crude product is to adopt solid phase synthesis
Method is dried obtained oxytocin precursor crude product through cracking, and HPLC purity is 60~90%.
3. preparation method as claimed in claim 1, it is characterised in that the structural formula of described oxytocin precursor crude product is Cys-
Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2Trifluoroacetate.
4. preparation method as claimed in claim 1, it is characterised in that described oxytocin precursor crude product solution is before oxytocin
Body crude product is dissolved in the 5g/L solution that the acetonitrile solution that concentration of volume percent is 5% is formed.
5. preparation method as claimed in claim 1, it is characterised in that described anti-phase cyclisation, anti-phase purification, anti-phase desalination are equal
It is to complete in the anti-phase elution process of a step.
6. preparation method as claimed in claim 1, it is characterised in that described anti-phase cyclisation, anti-phase purification, anti-phase desalination
Condition is as follows：Mobile phase A 1 is pure water, and A2 is the H of percent by volume 0.01~0.05%2O2PH be 7.5~9.0 NaOH it is water-soluble
Liquid, Mobile phase B is acetonitrile, and mobile phase C is described oxytocin precursor crude product solution, and flow velocity is 80~110mL/min, detects ripple
A length of 220nm；
Condition according to following table carries out online loading, eluting, and percentage ratio is percent by volume；
Collect the eluent that retention time is 50~65min and obtain oxytocin solution.
7. preparation method as claimed in claim 6, it is characterised in that described A2 is percent by volume 0.02~0.03%
H2O2PH is 7.5~9.0 NaOH aqueous solutions.
8. preparation method as claimed in claim 6, it is characterised in that described flow velocity is 100mL/min.
9. preparation method as claimed in claim 1, it is characterised in that the filling condition of described efficient liquid phase reverse-phase chromatography
It is as follows：Filler be Kromasil silica gel C18, aperture 10nm, 10 μm of particle diameter.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016072A (en) * | 2019-05-07 | 2019-07-16 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin |
| CN110041406A (en) * | 2019-05-07 | 2019-07-23 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin [+Gly] impurity |
| CN110066319A (en) * | 2019-05-07 | 2019-07-30 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin acetylation impurity |
| CN112175045A (en) * | 2020-10-28 | 2021-01-05 | 合肥亿帆生物制药有限公司 | Method for purifying oxytocin |
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| 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 |
| CN104086631A (en) * | 2014-07-10 | 2014-10-08 | 成都天台山制药有限公司 | Carbetocin and preparation method thereof |
| 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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| 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 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016072A (en) * | 2019-05-07 | 2019-07-16 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin |
| CN110041406A (en) * | 2019-05-07 | 2019-07-23 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin [+Gly] impurity |
| CN110066319A (en) * | 2019-05-07 | 2019-07-30 | 上海上药第一生化药业有限公司 | A kind of refining methd of oxytocin acetylation impurity |
| CN110066319B (en) * | 2019-05-07 | 2021-04-06 | 上海上药第一生化药业有限公司 | Method for refining oxytocin acetylation impurities |
| CN110041406B (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 [ + Gly ] impurity |
| 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 |
| CN112175045A (en) * | 2020-10-28 | 2021-01-05 | 合肥亿帆生物制药有限公司 | Method for purifying oxytocin |
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