CN105646669A

CN105646669A - Carbetocin purification method

Info

Publication number: CN105646669A
Application number: CN201610190022.8A
Authority: CN
Inventors: 徐红岩; 闫凤
Original assignee: Glbetter Biochemical (shanghai) Co Ltd
Current assignee: Glbetter Biochemical (shanghai) Co Ltd
Priority date: 2016-03-30
Filing date: 2016-03-30
Publication date: 2016-06-08

Abstract

The invention discloses a carbetocin purification method adopting reversed-phase high-performance liquid chromatography. The technical problems of high cost, operation complexity and low yield of carbetocin obtained by separation in the prior art are mainly solved. The method includes steps: 1) dissolving crude peptides obtained by solid synthesis into deionized water, taking a reverse phase silica gel column of octadecyl bonded silica gel as a stationary phase, taking 0.1 TFA aqueous solution as a phase A of a mobile phase and 0.1% TFA acetonitrile solution as a phase B of the mobile phase, performing gradient elution purification to collect a peptide solution of a target peak value; 2) adopting an HPLC method for conversion to obtain acetate; 3) subjecting a final high-purity peptide solution to pressure reduction, rotary evaporation, concentration, freezing and drying to obtain finished peptide powder. The carbetocin purification method is suitable for industrial carbetocin purification and is simple in operation, safe and low in cost.

Description

A kind of method of purifying Carbetocin

Technical field

The invention belongs to reverse-phase HPLC technique field, a kind of method especially relating to mass industrialization purifying Carbetocin.

Background technology

Carbetocin is the long-acting oxytocin nonapeptide analog with agonist characteristics of a kind of synthesis. Can be administered by Single-dose intravenous immediately after cesarotomy under epidural or lumbar anesthesia, to prevent uterus tension force not enough and postpartum hemorrhage. The clinic of carbetocin is similar with naturally-produced oxytocin with pharmacological property. As oxytocin, carbetocin is combined with the ocytocin receptor of uterine smooth muscle, causes the Rythmic contractions characteristic in uterus, on original contraction basis, increases its frequency and increases uterus tension force. Under non pregnant state, the ocytocin receptor content in uterus is very low, increases, reach peak during childbirth during gestation. Therefore nogestational uterus is not acted on by carbetocin, but the uterus of gestation and the uterus of harsh product are had effective uterine contraction effect.

Relative to oxytocin, the long action time of carbetocin, the uterine contraction produced therefrom cannot stop simply by terminating administration. So before baby gives birth to, no matter any reason all can not give carbetocin, including selectivity or drug-induced production. Whether, after intravenous injection or intramuscular injection carbetocin, uterus is shunk rapidly, can reach a clear and definite intensity in 2 minutes. The active function in uterus be may last about 1 hour by Single-dose intravenous injection carbetocin, is therefore enough to prevent the postpartum hemorrhage in harsh puerperal. After giving carbetocin puerperal, all long than oxytocin is in the frequency shunk with amplitude.

Research shows, when after cesarotomy under epidural or lumbar anesthesia immediately Single-dose intravenous give carbetocin 100 microgram, not enough at prevention uterus tension force and in reducing postpartum hemorrhage, carbetocin is substantially better than placebo. The restoration of old ways that carbetocin can also promote uterus is given puerperal early stage.

Postpartum hemorrhage is one of common severe complication of clinical obstetrics, is the first cause of current China maternal death. In four big reasons of postpartum hemorrhage, strength of uterine contraction is hemorrhage accounts for first place. Therefore, actively preventing and treating uterine contractions fatigue is hemorrhage, is the key reducing maternal mortality rate. At present China only only 2 pharmaceutical factories produce the medicament of carbetocins, first have to before producing medicament to guarantee to have and industrialization can produce the crude drug of carbetocin.Based on carbetocin also concrete very big application prospect clinically, there is significantly high Development volue, therefore, it is possible to industrialization obtain that highly purified carbetocin fine work just becomes very important.

Applicant once submitted 1 purifying Carbetocin patent application last year to, number of patent application is 201510107759.4, although this application yield is higher, but in purge process, introduce many inorganic sodiums, owing to its storage time is short, have the deposition of a lot of inorganic salts without flushing chromatographic column in time after purification is complete, cause the damage of chromatograph pillar, need often to change, and then too increase the cost of purification. And often rinse chromatographic column, purification time significantly can be extended again. Therefore on the basis of certain yield, how the problem that production cost is exactly the present invention and needs research is reduced better.

Summary of the invention

It is an object of the invention to provide a method being suitable to industrialization purifying Carbetocin, mainly solve prior art separation and obtain carbetocin cost height, the technical problem of troublesome poeration.

For achieving the above object, technical scheme is as follows:

A kind of method of purifying Carbetocin, comprises the steps:

1) by thick for solid phase synthesis gained peptide deionized water dissolving, fixing is reverse phase silica gel post mutually, mobile phase is: the TFA(trifluoroacetic acid containing 0.1% mass percentage concentration) aqueous solution is A phase, TFA acetonitrile solution containing 0.1% mass percentage concentration is B phase, carry out gradient elution purification, collect the peptide solution of purpose peak value;

2) the high-purity peptide solution after purification adopts HPLC turn the exchange of salt method and change into acetate;

3) final high-purity peptide solution vacuum rotary steam concentrated frozen is dried, obtain Powdered finished product.

The scheme being more highly preferred to is: gradient B%:15-65%, and detection wavelength is 220nm.

The scheme being more highly preferred to is: the described reverse phase silica gel that reverse phase silica gel post is octadecyl silane.

The scheme being more highly preferred to is: described HPLC turns salt method, and pH is aqueous acetic acid and the two phase liquid of trifluoroacetic acid aqueous solution of 3.0 ~ 5.0, and chromatographic column is the reverse phase silica gel post of octadecyl silane.

The invention has the beneficial effects as follows: a process being suitable to industrialization purifying Carbetocin of the proposition of the present invention, use reversed phase high-performance liquid chromatography purifying Carbetocin, and with HPLC turn salt method exchange turn salt, the HPLC purity fine peptide more than 98.5% can not only be obtained, and can mass production, certain yield basis reaches purity height, the requirement that cost is low.

Accompanying drawing explanation

Fig. 1 is embodiment 1 product chromatogram.

Fig. 2 is embodiment 2 product chromatogram.

Fig. 3 is embodiment 3 product chromatogram.

Detailed description of the invention

Embodiment 1

1. sample treatment: will the carbetocin deionized water dissolving (concentration of ordinary dissolution is about 500mg/ml) of synthesis gained, be that to collect filtrate after 0.45um membrane filtration standby with aperture;

2. purification

Purification condition: chromatographic column: the chromatographic column with 18 alkyl silica gel bonded silica gel for fixing phase, pillar diameter and length is: 10cm �� 30cm. mobile phase: A phase: the TFA aqueous solution containing 0.05% mass percentage concentration; B phase: the TFA acetonitrile solution containing 0.05% mass percentage concentration. Flow velocity: 100-120ml/min. Detection wavelength: 220nm. Gradient: B%:20-60%, 80-100min. Sample size is 5.1-10.8g;

Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 80% well rear loading, applied sample amount is sample solution.Linear gradient elution, collects purpose peak, places in receiving flask standby by the peptide solution gathered;

3. turn salt: fixing mutually for the reverse phase silica gel post of octadecyl silane with the deionized water rinsing of mass percentage concentration more than 90%, (substantially neutral) is 3.0 ~ 4.5 acetate aqueous solutions with the pH configured and trifluoroacetic acid aqueous solution two phase liquid balances chromatographic column again after rinsing well, after chromatographic column balance is thorough, starts loading turns salt, applied sample amount is the purpose peptide solution gathered, linear gradient elution: first with the acetate aqueous solution of 15-30min mass percentage concentration 80-90%, acetonitrile solution then through 30-60min mass percentage concentration 20-65%, collect whole target peaks,

4. by turning all peptide solutions of gained after salt, carrying out being evaporated to 1g/200ml, thickening temperature is less than 37 DEG C, and then lyophilization obtains the purity carbetocin more than 98.5%, and purification yield can obtain more than 60%. Referring to Fig. 1.

Embodiment 2

2. purification

Purification condition: chromatographic column: the chromatographic column with 18 alkyl silica gel bonded silica gel for fixing phase, pillar diameter and length is: 10cm �� 30cm. mobile phase: A phase: the TFA aqueous solution containing 0.1% mass percentage concentration; B phase: the TFA acetonitrile solution containing 0.1% mass percentage concentration. Flow velocity: 100-120ml/min. Detection wavelength: 220nm. Gradient: B%:15-55%, 80-100min. Sample size is 9.0-11.7g;

Purge process: rinse the acetonitrile of chromatographic column mass percentage concentration more than 80% well rear loading, applied sample amount is sample solution. Linear gradient elution, collects purpose peak, places in receiving flask standby by the peptide solution gathered;

3. turn salt: fixing mutually for the reverse phase silica gel post of octadecyl silane with the deionized water rinsing of mass percentage concentration more than 90%, (substantially neutral) is 3.5 ~ 5.0 acetate aqueous solutions with the pH configured and trifluoroacetic acid aqueous solution two phase liquid balances chromatographic column again after rinsing well, after chromatographic column balance is thorough, starts loading turns salt, applied sample amount is the purpose peptide solution gathered, linear gradient elution: first with the acetate aqueous solution of 15-30min mass percentage concentration 80-90%, acetonitrile solution then through 30-70min mass percentage concentration 15-60%, collect whole target peaks,

4. by turning all peptide solutions of gained after salt, carrying out being evaporated to 1g/200ml, thickening temperature is less than 37 DEG C, and then lyophilization obtains the purity carbetocin more than 98.5%, and purification yield can obtain more than 70%. Referring to Fig. 2.

Embodiment 3

1. sample treatment: will the carbetocin deionized water dissolving (concentration of ordinary dissolution is about 1000mg/ml) of synthesis gained, be that to collect filtrate after 0.45um membrane filtration standby with aperture;

2. purification

Purification condition: chromatographic column: the chromatographic column with 18 alkyl silica gel bonded silica gel for fixing phase, pillar diameter and length is: 15cm �� 50cm. mobile phase: A phase: the TFA aqueous solution containing 0.15% mass percentage concentration; B phase: the TFA acetonitrile solution containing 0.15% mass percentage concentration. Flow velocity: 150-180ml/min. Detection wavelength: 220nm. Gradient: B%:15-65%, 100-120min. Sample size is 18.0-20.0g;

3. turn salt: fixing mutually for the reverse phase silica gel post of octadecyl silane with the deionized water rinsing of mass percentage concentration more than 90%, (substantially neutral) is 3.5 ~ 5.5 acetate aqueous solutions with the pH configured and trifluoroacetic acid aqueous solution two phase liquid balances chromatographic column again after rinsing well, after chromatographic column balance is thorough, starts loading turns salt, applied sample amount is the purpose peptide solution gathered, linear gradient elution: first with the acetate aqueous solution of 10-30min mass percentage concentration 60-80%, acetonitrile solution then through 50-100min mass percentage concentration 15-65%, collect whole target peaks,

4. by turning all peptide solutions of gained after salt, carrying out being evaporated to 1g/200ml, thickening temperature is less than 37 DEG C, and then lyophilization obtains the purity carbetocin more than 98.5%, and purification yield can obtain more than 75%. Referring to Fig. 3.

In sum; low with the method purifying Carbetocin cost; operation is simple; for industrialized purification carbetocin; safety coefficient is high; highly purified fine work can be obtained again while method is simple and easy to get, be strictly a good purification process, additionally be suitably modified on the basis of this patent and will need to be protected.

Claims

1. a method for purifying Carbetocin, adopts reversed phase high-performance liquid chromatography, it is characterized in that comprising the steps:

1) by thick for solid phase synthesis gained peptide deionized water dissolving, fixing is reverse phase silica gel post mutually, mobile phase has biphase, trifluoroacetic acid aqueous solution containing 0.1% mass percentage concentration is A phase, trifluoroacetic acid acetonitrile solution containing 0.1% mass percentage concentration is B phase, carry out gradient elution purification, collect the peptide solution of purpose peak value;

2. the method for purifying Carbetocin according to claim 1, is characterized in that: described gradient elution, the value range B%=15-65% of mass percentage concentration, and detection wavelength is 220nm.

3. the method for purifying Carbetocin according to claim 1, is characterized in that: described reverse phase silica gel post is octadecylsilane chemically bonded silica.

4. the method for purifying Carbetocin according to claim 1, is characterized in that: described HPLC turns salt method, and pH is aqueous acetic acid and the two phase liquid of trifluoroacetic acid aqueous solution of 3.0 ~ 5.0, and chromatographic column is the reverse phase silica gel post of octadecylsilane chemically bonded silica.