CN110016072A - A kind of refining methd of oxytocin - Google Patents
A kind of refining methd of oxytocin Download PDFInfo
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- CN110016072A CN110016072A CN201910375969.XA CN201910375969A CN110016072A CN 110016072 A CN110016072 A CN 110016072A CN 201910375969 A CN201910375969 A CN 201910375969A CN 110016072 A CN110016072 A CN 110016072A
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- oxytocin
- crude product
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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 kind of refining methd of oxytocin, which includes the following steps: oxytocin crude product solution successively carried out reverse phase enrichment using efficient liquid phase RP chromatography, reverse phase turns salt, reverse phase purifying；Oxytocin crude product solution is that the reduced form oxytocin crude product solution of synthesis in solid state obtains after aoxidizing；The filler of efficient liquid phase RP chromatography is super resistance to water packing.The present invention is enriched with online reverse phase, reverse phase turns salt, polypeptide sterling is made in reverse phase purifying one-step method, optimize production technology, the mobile phase that wherein column equilibration, loading were enriched with and turned salt phase is aqueous solution, and environment friendly and pollution-free, the waste liquid of outflow can directly carry out sewage treatment and recycle.
Description
Technical field
The present invention relates to a kind of refining methds of oxytocin.
Background technique
Oxytocin, also known as oxytocins, the entitled Oxytocin of English, structural formula:
Molecular formula are as follows: C43H66N12O12S2, molecular weight 1007.19
Oxytocin is used for induced labor, hastens parturition, postpartum and the post-abortion uterine hemorrhage because caused by uterine atony or contracting abdomen are bad；
Understand placenta reserve function (oxytocins enrages test)；Collunarium can promote milk ejection.Oxytocin energy indirect stimulation uterine smooth muscle is received
Contracting simulates the uterine contraction effect of normal labor, leads to cervical dilatation, uterus to the reaction of oxytocin during pregnancy by
It is cumulative to add, peak is reached when mature.Oxytocin can also pierce mammotropic smooth muscle contraction, facilitate milk and be discharged from breast, but simultaneously
The galactosis amount of mammary gland is not increased.
Having listed the common purification process of polypeptide drugs mostly uses preparative high performance liquid chromatography at present, which is
Obtain the most efficient method of high-purity polypeptide target molecule.General polypeptide drugs purifying preparation process design is first mesolow color
Spectrum enriching target polypeptides, then high pressure chromatographic refining, it is contemplated that target polypeptides oxytocin molecule amount about 1kDa, without suitable
Molecular sieve gel column (its applied sample amount is small, and flow velocity is low, and treating capacity is small, relatively be suitble to molecular weight be greater than 10kDa albumen desalination) or
Ultrafiltration membrane selection.And common separation method has molecular sieve chromatography, ion-exchange chromatography and hydrophobic in mesolow chromatography
Interaction chromatography method, the partial size for the filler used in these chromatographic processes are empty usually from tens microns to several hundred microns etc.
Gap size is mostly several hundred nanometers etc., is unable to get the target polypeptides of high-purity.The contracting being cyclized using synthesis in solid state+dilution
Palace element crude product solution concentration is diluter, when being purified using general reverse-phase chromatographic column, only just generates during loading
The processing cost of a large amount of organic liquid waste, dangerous waste is very high.A kind of method for effectively preparing polypeptide salt bulk drug is also lacked now,
Therefore still there is an urgent need to develop the cost-effective techniques of new suitable purifying low concentration polypeptide and salt.
Chinese patent literature CN106674332A discloses a kind of preparation method of oxytocin.The preparation method includes following
Step: oxytocin precursor crude product solution is successively carried out by reverse phase cyclisation, reverse phase purifying, reverse phase using efficient liquid phase RP chromatography
Desalination；The filler of efficient liquid phase RP chromatography is silica gel C18；The oxytocin precursor crude product is containing two free mercaptos
The oxytocin precursor crude product of base.Use the oxytocin precursor crude product of reduced form for starting material in the preparation process of the patent, no
The largely mobile phase containing organic solvent is needed when being only cyclized on a column and is being purified and is being turned also to generate in salt step
A large amount of organic dangerous waste liquid, the processing cost of subsequent waste liquid are larger.
Chinese patent literature CN1990501A discloses a kind of method of synthesis in solid state oxytocin, and this method is with Rink
Amide or Rink MBHA resin is that starting material is gradually synthesized using TBTU/HOBt or HABU/HOBt as condensing agent；Then it cuts
It cuts, with air or with hydrogen peroxide (10-3~10-4Mol/L) solution is aoxidized, Yi Fasheng intermolecular reaction generate it is linear or
The shortcomings that cricoid dimerization and polymer, this method, is that the oligomerization between peptide molecule easily causes cyclic peptide yield and purity
It is serious to reduce, and in the preparation process of this method mobile phase be containing organic solvent, the danger treatment cost of waste liquor of generation it is high-leveled and difficult with
It recycles.
Summary of the invention
It is organic danger technical problem to be solved by the present invention lies in overcoming the dangerous waste liquid measure generated in the prior art big
Waste liquid, caused by treatment cost of waste liquor it is big, uneconomic defect, and provide a kind of refining methd of oxytocin.Of the invention
The waste liquid that the refining methd of oxytocin generates during purification of target product is largely that waste water can be straight through sewage treatment
Use is taken back, it is economic and environment-friendly.
The present invention is to solve above-mentioned technical problem by the following technical programs:
The present invention provides a kind of refining methds of oxytocin comprising following step:
Oxytocin crude product solution is successively carried out by reverse phase enrichment using efficient liquid phase RP chromatography, reverse phase turns salt, reverse phase
Purifying；
The filler of the efficient liquid phase RP chromatography is super resistance to water packing；
The reverse phase is enriched with, reverse phase turns salt, reverse phase purifying is completed in a step reverse phase elution process, and described is anti-
Mutually enrichment, the condition that reverse phase turns salt, reverse phase purifies are as follows:
Wherein, mobile phase A is the acetic acid/water that percent by volume is 0.005~0.1%, and Mobile phase B is that percent by volume is
0.005~0.1% acetic acid/acetonitrile, sample C1 are the oxytocin crude product solution, and mobile phase C2 is 5~50mM NH4Ac-
NH4OH aqueous solution, the pH of the mobile phase C2 are 7.0~9.0, and flow velocity is 80~100ml/min；
Collecting retention time is the eluent of 95~105min up to oxytocin solution.
In the present invention, the oxytocin crude product solution be synthesis in solid state reduced form oxytocin crude product through dissolution, dilution,
It aoxidizes and obtains.
Specific preparation process is as follows for the oxytocin crude product solution: being that starting is former with Rink Amide MBHA resin
Material, using fmoc-protected amino acid as monomer, using HOBt/DIC as condensing agent, successively connects amino acid one by one；Peptide examination is cut in addition
Agent carries out cutting peptide, and methyl tertiary butyl ether(MTBE) is added and is precipitated, reduced form oxytocin crude product is obtained；Reduced form oxytocin crude product is used
50% acetic acid/water dissolves, then is diluted with water, as reduced form oxytocin crude product solution；With alkaline matter by the reduced form
The pH of oxytocin crude product solution is adjusted to 7.0-9.0, and the hydrogen peroxide that concentration is 30% is added and is aoxidized, wherein every gram of reduced form
Oxytocin crude product adds the hydrogen peroxide of 0.5ml30%, obtains oxidized form oxytocin crude product solution, as oxytocin crude product solution.
Wherein, 50% acetic acid/water can adequately dissolve the reduced form oxytocin crude product.
Wherein, the concentration of the reduced form oxytocin crude product solution is 0.1~4mg/ml, preferably 0.5~2mg/
ml。
Wherein, the described peptide reagent of cutting is that this field is conventional, preferably the volume ratio TFA/TIS/ that is 90:7.5:2.5
H2O。
Wherein, the alkaline matter is that this field is conventional, preferably NaOH.
In the present invention, the structural formula of oxytocin is in the oxytocin crude product solutionSolvent in the oxytocin crude product solution is containing trifluoroacetic acid
With the aqueous solution of acetic acid.
In the present invention, the super resistance to water packing isThe super resistance to water packing of ODS-AQ.
In the present invention, the aperture of the super resistance to water packing is 7~10nm, and partial size is 10 μm.
In a certain better embodiment, with Load&Lock dynamic axial compression and static locking technology, filler isThe super resistance to water packing of ODS-AQ, aperture 10nm, 10 μm of partial size, being filled to column bed pressure is 1000psi, is used
Varian chromatography loading system, 300g dry powder-shapedThe super resistance to water packing of ODS-AQ, the stirring homogenate of 600mL isopropanol
Afterwards, the Load&Lock4002 for pouring into internal diameter 50mm prepares column, compression ratio 1.5:1, carrier gas N2, adjust the nebulizer gas pressure
So that oil pressure meter pressure is 1500psi, dynamic axial compression to column bed height 25cm is enriched with as reverse phase, reverse phase turns salt and anti-
Column is prepared used in phase purification schemes.
In the present invention, the Detection wavelength of the efficient liquid phase RP chromatography is 220nm.
In the present invention, the mobile phase A is the acetic acid/water solution that percent by volume is 0.02~0.05%；
And/or the Mobile phase B is acetic acid/acetonitrile that percent by volume is 0.02~0.05%；
And/or the mobile phase C2 is 10~20mM NH4Ac-NH4OH aqueous 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
It goodly is 70%~80%；
And/or the eluent that the collection retention time is 98~101min is oxytocin solution.
In the present invention, during 50~51min, the eluent is changed to mobile phase C2 by sample C1 completely；?
During 71~72min, the eluent is changed to mobile phase A by mobile phase C2 completely.According to this field routine, when above-mentioned
Between section should not be construed as the restriction to elution requirement, length of time can be carried out according to the difference of high performance liquid chromatograph producer model
Corresponding adjustment.
In the present invention, in 115~116min after step (5), at the uniform velocity from 80% mobile phase A by the eluent
Be reduced to 50% mobile phase A, accordingly at the uniform velocity increase Mobile phase B to 50%, kept in 116~125min 50% mobile phase A+
50% Mobile phase B elution, to achieve the purpose that clean chromatographic column.
Wherein, reverse phase enrichment is elution step (1), and it is step (2)~(3) that the reverse phase, which turns salt, specifically,
Step (2) is with weak base NH4Ac-NH4OH removes the process of the trifluoroacetic acid root in oxytocin crude product, and step (3) is removal step
(2) process of the ammonium ion in, the reverse phase purifying is step (4) and (5), wherein step (4) is to remove weaker suction
The process of attached impurity.
In the present invention, the conversion rate of eluent described in the elution step (4) and (5) is one and at the uniform velocity changes
Process, the rate at the uniform velocity changed described in the elution step (4) be 2%B/min, i.e., per minute in former eluent
On the basis of increase by 2% described in Mobile phase B, while it is corresponding reduce 2% described in mobile phase A；Institute in the elution step (5)
Stating the rate that at the uniform velocity changes is 0.333%B/min, i.e., increase by 0.333% on the basis of former eluent per minute described in stream
Dynamic phase B, while mobile phase A described in corresponding reduction 0.333%.
Oxytocin is a kind of peptide material, unstable under high ph conditions, degradable, especially under alkali environment, the present invention
Integrated survey turns pH and the time of eluting salt, with guarantee to reduce turn salt during sample destruction and loss.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
(1) method that the present invention uses on-line preconcentration, using the super water resistance and absorption property of filler, by polypeptide crude product
It is adsorbed onto stationary phase and is enriched with, polypeptide and reverse phase filler hydrophobic binding.It is laggard that mobile phase can be directly converted after on-line preconcentration
The purifying of row gradient elution, obtains final sterling, is suitble to continuous production.
(2) present invention has innovatively used reverse phase absorption enrichment, has turned the obtained polypeptide sterling of salt, desalination one-step method, optimization
Production technology is suitble to industrialization continuous production.
(3) more recent application of the water-fast filler of excess of export is designed, column equilibration stage, reverse phase concentration stage and reverse phase turn salt phase
Eluent is aqueous solution, environment friendly and pollution-free, and efflux is immediately discharged to Sewage Disposal, the recoverable after simple process,
The yield of dangerous waste liquid, economical environment-protective greatly reduces in relatively traditional preparation process.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Resistance to water packing used in embodiment is purchased from Suzhou Nano-Micro Bio-technology Co., Ltd.ODS-AQ is super
Resistance to water packing, aperture 10nm, 10 μm of partial size.
HPLC method detects oxytocin crude product and after purification product solution purity:
Instrument: 1200 high performance liquid chromatograph of Agilent
Splitter: Waters XBridge-C18,4.6 × 150mm, 5 μm
Mobile phase: A is 50% acetonitrile/water solution of percent by volume, and B is 0.02M KH2PO4PH3.0 aqueous solution, flow velocity are
1.0ml/min, Detection wavelength 220nm, room temperature detection, gradient see the table below shown, and percentage 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 | 28~28.1min | 100%A → 35%A+65%B |
| 5 | 28.1~32min | 35%A+65%B |
In following embodiments, the oxytocin crude product solution be synthesis in solid state reduced form oxytocin crude product through dissolution,
Dilution is aoxidized and is obtained.The solid-phase synthesis includes the following steps: using Rink Amide MBHA resin as starting material, with
Fmoc-protected amino acid successively connects amino acid using HOBt/DIC as condensing agent for monomer one by one；Peptide reagent progress is cut in addition
Peptide is cut, methyl tertiary butyl ether(MTBE) is added and is precipitated, reduced form oxytocin crude product is obtained.It is described that cut peptide reagent be volume ratio is 90:
The TFA/TIS/H of 7.5:2.52O.Described is dissolved as being dissolved with the acetic acid/water solution that percent by volume is 50%.It is described
Be diluted to be diluted with water.The pH of the reduced form oxytocin crude product solution is adjusted to by described being oxidized to alkaline matter
7.0-9.0 is added the hydrogen peroxide that percent by volume is 30% and carries out oxidation process.The dosage of the hydrogen peroxide be 0.5mL/1g also
Prototype oxytocin crude product.The alkaline matter is NaOH.
1 internal diameter 50mm L&L4002 of embodiment prepares column filling:
With Load&Lock dynamic axial compression and static locking technology, filler isODS-AQ, aperture
10nm 10 μm of partial size, is filled to column bed pressure 1000psi, using Varian chromatography loading system, 300g dry powder filler, 600ml
After isopropanol stirring homogenate, pours into internal diameter 50mm L&L4002 and prepare column, compression ratio 1.5:1, carrier gas N2, adjust carrier gas pressure
Power make oil pressure meter pressure be 1500psi, dynamic axial compression to column bed height 25cm, as reverse phase enrichment, reverse phase turn salt and
Column is prepared used in reverse phase purification schemes.
The reverse phase enrichment of 2 oxytocin crude product solution of embodiment, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: 1 self-chambering of embodiment prepares column Load&Lock400250 × 250mm,ODS-AQ10μ
m10nm
The structural formula of oxytocin isReduced form oxytocin crude product
The concentration of reduced form oxytocin crude product is 1mg/ml in solution, and the solvent in oxytocin crude product solution is containing trifluoroacetic acid and acetic acid
Aqueous solution.
Mobile phase A is that percent by volume is 0.02% acetic acid/water, and Mobile phase B is that percent by volume is 0.02% acetic acid/second
Nitrile, sample C1 are oxytocin crude product solution, and the HPLC purity according to the oxytocin crude product solution of HPLC method measurement is 71.23%,
Mobile phase C2 is the NH of 10mM4Ac-NH4OH pH7.5 aqueous solution.
The reverse phase enrichment of the present embodiment, reverse phase turns salt and reverse phase purification condition is as follows: flow velocity 100ml/min, 220nm inspection
It surveys, purifying gradient see the table below shown, and percentage is percent by volume.
It collects the eluent that retention time is 95~105min and obtains oxytocin solution.The contracting palace measured according to HPLC method
The purity of plain HPLC is 99.63%；Collect the purity for the oxytocin HPLC that the eluent that retention time is 98~101min obtains
It is 99.85%.
What the present embodiment one-step method completed oxytocin crude product solution turns salt and purifying, elution step (1)~(3) stage
Eluent be aqueous solution, environment friendly and pollution-free, efflux is immediately discharged to Sewage Disposal, after simple process can be recycled benefit
With the yield of dangerous waste liquid, economical environment-protective greatly reduces in relatively traditional preparation process.
The reverse phase enrichment of 3 oxytocin crude product solution of embodiment, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: 1 self-chambering of embodiment prepares column Load&Lock400250 × 250mm,ODS-AQ10μ
m10nm
The structural formula of oxytocin isReduced form oxytocin crude product
The concentration of reduced form oxytocin crude product is 1.5mg/ml in solution, and the solvent in oxytocin crude product solution is containing trifluoroacetic acid and second
The aqueous solution of acid.
Mobile phase A is that percent by volume is 0.05% acetic acid/water, and Mobile phase B is that percent by volume is 0.05% acetic acid/second
Nitrile, sample C1 are oxytocin crude product solution, and the purity according to the oxytocin crude product solution HPLC of HPLC method measurement is 71.03%,
Mobile phase C2 is the NH of 20mM4Ac-NH4OH pH8.5 aqueous solution.
The reverse phase enrichment of the present embodiment, reverse phase turns salt and reverse phase purification condition is as follows: flow velocity 100ml/min, 220nm inspection
It surveys, purifying gradient see the table below shown, and percentage is percent by volume.
It collects the eluent that retention time is 95~105min and obtains oxytocin solution.The contracting palace measured according to HPLC method
The purity of plain HPLC is 99.43%；Collect the purity for the oxytocin HPLC that the eluent that retention time is 98~101min obtains
It is 99.82%.
The reverse phase enrichment of 4 oxytocin crude product solution of embodiment, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: 1 self-chambering of embodiment prepares column Load&Lock400250 × 250mm,ODS-AQ10μ
m10nm
The structural formula of oxytocin isReduced form oxytocin crude product
The concentration of reduced form oxytocin crude product is 0.8mg/ml in solution, and the solvent in oxytocin crude product solution is containing trifluoroacetic acid and second
The aqueous solution of acid.
Mobile phase A is that percent by volume is 0.05% acetic acid/water, and Mobile phase B is that percent by volume is 0.05% acetic acid/second
Nitrile, sample C1 are oxytocin crude product solution, and the purity according to the oxytocin crude product solution HPLC of HPLC method measurement is 70.46%,
Mobile phase C2 is the NH of 20mM4Ac-NH4OH pH7.5 aqueous solution.
The reverse phase enrichment of the present embodiment, reverse phase turns salt and reverse phase purification condition is as follows: flow velocity 100ml/min, 220nm inspection
It surveys, purifying gradient see the table below shown, and percentage is percent by volume.
Collecting retention time is the eluent of 95~105min up to oxytocin solution.The oxytocin measured according to HPLC method
HPLC purity be 99.54%；Collecting the HPLC purity of oxytocin that the eluent that retention time is 98~101min obtains is
99.87%.
The Mass Spectrometer Method of 5 oxytocin of embodiment
It is obtained using Waters micromass ZQ substance level four bars electrospray ionization mass spectrum (ESI-MS) measurement embodiment 2,3 and 4
The oxytocin arrived, test condition are as follows: the source electrospray ionisation (ESI) is used, in positive ion electrospray from being analyzed by mass spectrometry under mode, hair
Tubule ionization voltage 3.0kV samples orifice potential 35kv；115 DEG C of ion source temperature, 350 DEG C of desolventizing temperature, desolventizing nitrogen
Flow velocity 700L/h, taper hole blowback nitrogen 50L/h, 50.0~1500m/z of level four bars surface sweeping range.
The result of detection are as follows: molecular ion peak [M+H]+Mass-to-charge ratio (m/z) is 1007.45, leading ion fragment peak [M+
2H]2+Mass-to-charge ratio (m/z) is 504.22, and all meeting the theoretical value i.e. molecular weight of oxytocin is 1007.19.
Claims (10)
1. a kind of refining methd of oxytocin, which is characterized in that it includes the following steps: to use efficient liquid phase RP chromatography will
Oxytocin crude product solution successively carries out reverse phase enrichment, reverse phase turns salt, reverse phase purifying；
The filler of the efficient liquid phase RP chromatography is super resistance to water packing；
The reverse phase is enriched with, reverse phase turns salt, reverse phase purifying is completed in a step reverse phase elution process, and the reverse phase is rich
It is as follows that collection, reverse phase turn salt, the condition of reverse phase purifying:
Wherein, mobile phase A is the acetic acid/water that percent by volume is 0.005~0.1%, and Mobile phase B is that percent by volume is
0.005~0.1% acetic acid/acetonitrile, sample C1 are the oxytocin crude product solution, and mobile phase C2 is 5~50mM NH4Ac-
NH4OH aqueous solution, the pH of the mobile phase C2 are 7.0~9.0, and flow velocity is 80~100ml/min；
Collecting retention time is the eluent of 95~105min up to oxytocin solution.
2. the refining methd of oxytocin as described in claim 1, it is characterised in that: the oxytocin crude product solution is solid phase
The reduced form oxytocin crude product of synthesis through dissolution, dilution, oxidation and obtain.
3. the refining methd of oxytocin as described in claim 1, it is characterised in that: the reduced form oxytocin crude product is through molten
It solves, dilute to obtain reduced form oxytocin crude product solution, reduced form oxytocin crude product in the reduced form oxytocin crude product solution
Concentration is 0.1~4mg/ml, preferably 0.5~2mg/ml；The solvent for dissolving the reduced form oxytocin crude product is volume hundred
Divide the acetic acid/water solution than being 50%.
4. the refining methd of oxytocin as described in claim 1, it is characterised in that: oxytocin in the oxytocin crude product solution
Structural formula beSolvent in the oxytocin crude product solution is
Aqueous solution containing trifluoroacetic acid and acetic acid.
5. the refining methd of oxytocin as described in claim 1, it is characterised in that: the super resistance to water packing is
The super resistance to water packing of ODS-AQ.
6. the refining methd of oxytocin as described in claim 1, it is characterised in that: the aperture of the super resistance to water packing be 7~
10nm, partial size are 10 μm.
7. the refining methd of oxytocin as described in claim 1, it is characterised in that: the efficient liquid phase RP chromatography
Detection wavelength is 220nm.
8. the refining methd of oxytocin as described in claim 1, it is characterised in that: the mobile phase A is percent by volume
For 0.02~0.05% acetic acid/water solution；
And/or the Mobile phase B is acetic acid/acetonitrile that percent by volume is 0.02~0.05%；
And/or the mobile phase C2 is 10~20mM NH4Ac-NH4OH aqueous 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
It is 70%~80%；
And/or the eluent that the collection retention time is 98~101min is oxytocin solution.
9. the refining methd of oxytocin as described in claim 1, it is characterised in that: during 50~51min, by eluent C1
It is changed to C2 completely, during 71~72min, eluent C2 is changed to A completely.
10. the refining methd of oxytocin as described in claim 1, it is characterised in that: the elution step (4) and elution step
Suddenly the conversion rate of the eluent in (5) is the process at the uniform velocity changed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175045A (en) * | 2020-10-28 | 2021-01-05 | 合肥亿帆生物制药有限公司 | Method for purifying oxytocin |
| 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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