CN103386118A - Method for controlling organic solvent residue in thymosin alpha 1 microspheres - Google Patents
Method for controlling organic solvent residue in thymosin alpha 1 microspheres Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 84
- 239000003960 organic solvent Substances 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 69
- 108010078233 Thymalfasin Proteins 0.000 title claims abstract description 30
- 102400000800 Thymosin alpha-1 Human genes 0.000 title claims abstract description 30
- NZVYCXVTEHPMHE-ZSUJOUNUSA-N thymalfasin Chemical compound CC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NZVYCXVTEHPMHE-ZSUJOUNUSA-N 0.000 title claims abstract description 30
- 229960004231 thymalfasin Drugs 0.000 title claims abstract description 30
- 238000003756 stirring Methods 0.000 claims abstract description 46
- 239000000839 emulsion Substances 0.000 claims abstract description 37
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 claims 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 abstract description 39
- 230000008569 process Effects 0.000 abstract description 38
- 101800001530 Thymosin alpha Proteins 0.000 abstract 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 46
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- 238000002156 mixing Methods 0.000 description 20
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- 239000011806 microball Substances 0.000 description 6
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 5
- WBJINCZRORDGAQ-UHFFFAOYSA-N formic acid ethyl ester Natural products CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 description 5
- 238000004817 gas chromatography Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 4
- 229930195725 Mannitol Natural products 0.000 description 4
- 108010046075 Thymosin Proteins 0.000 description 4
- 102000007501 Thymosin Human genes 0.000 description 4
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 4
- 239000004205 dimethyl polysiloxane Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000000834 fixative Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 235000010355 mannitol Nutrition 0.000 description 4
- 239000000594 mannitol Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 4
- 229920000747 poly(lactic acid) Polymers 0.000 description 4
- 239000004626 polylactic acid Substances 0.000 description 4
- 229920001296 polysiloxane Polymers 0.000 description 4
- 230000000630 rising effect Effects 0.000 description 4
- 230000002992 thymic effect Effects 0.000 description 4
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000009477 glass transition Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000009413 insulation Methods 0.000 description 3
- -1 polysiloxane Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010013786 Dry skin Diseases 0.000 description 2
- YNQLUTRBYVCPMQ-UHFFFAOYSA-N Ethylbenzene Chemical compound CCC1=CC=CC=C1 YNQLUTRBYVCPMQ-UHFFFAOYSA-N 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 2
- IVSZLXZYQVIEFR-UHFFFAOYSA-N m-xylene Chemical group CC1=CC=CC(C)=C1 IVSZLXZYQVIEFR-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- DQCKKXVULJGBQN-XFWGSAIBSA-N naltrexone Chemical compound N1([C@@H]2CC3=CC=C(C=4O[C@@H]5[C@](C3=4)([C@]2(CCC5=O)O)CC1)O)CC1CC1 DQCKKXVULJGBQN-XFWGSAIBSA-N 0.000 description 2
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- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- JECYNCQXXKQDJN-UHFFFAOYSA-N 2-(2-methylhexan-2-yloxymethyl)oxirane Chemical compound CCCCC(C)(C)OCC1CO1 JECYNCQXXKQDJN-UHFFFAOYSA-N 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
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- 235000002597 Solanum melongena Nutrition 0.000 description 1
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- 230000003308 immunostimulating effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a method for controlling organic solvent residue in thymosin alpha 1PLGA microspheres. The method comprises the following steps: stirring a prepared thymosin alpha 1PLGA microsphere emulsion or multiple emulsion under normal temperature and normal pressure at a speed of 500-1200rpm for 3 hours; stirring the emulsion or multiple emulsion for 2-18 hours under the conditions of vacuum degree of -0.035MP to -0.096MP, a temperature of 26-35 DEG C and rotating speed of 50-800 rpm. The method for controlling organic solvent residue in thymosin alpha 1 microspheres provided by the invention is efficient, controllable, stable in process, safe, reasonable and capable of effectively controlling the residue problem of such organic solvents as dichloromethane and keeping the integral forms of the microspheres at the same time.
Description
The application is to be December in 2008 23 days the applying date, and application number is CN200810240539.9, and denomination of invention is divided an application for " controlling the method for residue of organic solvent in thymic peptide alpha 1 microballoon " patent application.
Technical field
The present invention relates to a kind of preparation technology of pharmaceutical preparation, be specifically related to control the method for Determination of Residual Organic Solvents in Thymosin alpha 1 PLGA microsphere, belong to drug world.
Background technology
Thymosin alpha 1 (thymosin alpha1 is called for short T α 1) is a kind of polypeptide with immunologic function, and multiplex in immunostimulant or treatment viral hepatitis clinically, its aminoacid sequence is:
ΑC-Ser-Αsp-Αlα-Αlα-Vαl-Αsp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Α sp-Leu-lys-Glu-Lys-Lys-Glu-Vαl-Vαl-Glu-Glu-Αlα-Glu-Αsn_-OH
Molecular formula: C
129H
215N
33O
55
Molecular weight: 3108.37
Existing Thymosin alpha 1 preparation, because short treatment time of half-life is long, needs frequent drug administration, has the shortcomings such as the poor and bioavailability of patient compliance is low.Adopt at present microball preparation by thymosin T α 1 is wrapped in spheroid, be prepared into sustained-release micro-spheres, slow, controlled release thymosin T α 1, make the patient, by T α 1 microball preparation of injection, reach longer therapeutic purposes.
It is framework material that slow release microphere for injection often adopts poly lactic coglycolic acid (PLGA), parcel thymosin T α 1, in preparation process, need with an organic solvent, because organic solvent has certain harm to human body, environment, studies confirm that their accumulation in human body easily bring out and accelerate generation and the development of various diseases, be to ensure drug quality and drug safety, especially, for injection preparation, must control organic solvent residual.
Existing report about Thymosin alpha 1 PLGA microsphere, remove organic solvent by direct lyophilization or the stirring at normal temperature volatilization method in later stage.As (researchs of Thymosin alpha 1 release injectable microsphere such as Zhu Yan, Acta Pharmaceutica Sinica, vol(42) 2:211～215) report: use immediately distilled water diluting after preparing colostrum that T α 1 emulsifying makes, emulsion, stirring at low speed (400r/min-) 4h, centrifugal collection microsphere, distilled water wash 3 times, lyophilization 24h makes microsphere.ZL02136181.9 discloses the preparation method of Thymosin alpha 1 microball preparation, adopts stirring at normal temperature that organic solvent is volatilized.
Wrapped up by PLGA due to organic solvent or with PLGA adhesion or absorption, adopt said method not shoot from microsphere fully, in spheroid, the residual quantity of the organic solvent such as dichloromethane still remains on a relatively high level, can't further reduce again.
Also there is bibliographical information to remove organic solvent by the mode of rising temperature, how bright grade (preparation of thymosin polylactic acid microsphere and Release Performance research, Chinese Journal of Pharmaceuticals, 2006, vol37 (3): 178～180) report Thymosin alpha 1 PLA microsphere, 45 ℃ of stirring microspheres, volatilization dichloromethane, after washing, filtration, 60 ℃ of dried overnight; (heating under diminished pressure is removed residual dichloromethane in naltrexone microsphere to Luan Hansen, Chinese Journal of Pharmaceuticals, 2005, vol36 (9): 545～547) report: the naltrexone PLGA microsphere that obtains after the lyophilization of learning from else's experience is being decompressed to 100pa, reducing pressure under the high temperature of 50～65 ℃, it is dry to heat up, only under 65 ℃, the condition of 100pa through 48 hours, in microsphere, Determination of Residual Organic Solvents can be down to 0.04%, to release phenomenon serious but microsphere is prominent; Be decompressed to 100pa, 50～60 ℃ of dryings 24～48 hours and 100pa, the residual quantity of 65 ℃ of dryings dichloromethane after 24 hours still maintains between 0.17～1.1%, not only can not guarantee the quality of microsphere, can not effectively reduce the content of organic solvent.The temperature of above-mentioned bibliographical information is all more than 45 ℃, because the glass transition temperature of PLGA is generally 45～55 ℃; Micromolecule organic solvent such as trace dichloromethane etc. is with after PLGA mixes, and the glass transition temperature of PLGA can change or reduce (Zheng Qiaodong etc., medicine control releasing research and utilization, Zhejiang chemical industry, 2003, vol (34) 5:26～29).In microspheres, if temperature is close to or higher than the glass transition temperature of PLGA, PLGA elasticity increases, and when microsphere came in contact and collides, microsphere easily occurs to merge or spheroid breaks and causes Thymosin alpha 1 seepage or outflow; Temperature due to emulsion raises simultaneously, the microsphere spheroid exists and forms duct, and the trend that merges occurs the duct through colliding a plurality of microspheres, causes microsphere stick together, merge or break, make the prominent efficiency of releasing seriously or losing slow controlled release of this microball preparation, can not be as medicinal.Therefore, only by the rising temperature, the residual of organic solvent can not be reduced, the quality of microball preparation can not be guaranteed.
Therefore, need organic solvent residual in a kind of effective reduction Thymosin alpha 1 PLGA microsphere, can keep the method for microsphere intact form simultaneously.
Summary of the invention
Technical scheme of the present invention has been to provide a kind of method of controlling organic solvent residual in Thymosin alpha 1 PLGA microsphere.
The invention provides a kind of method of controlling organic solvent residual in Thymosin alpha 1 PLGA microsphere, it be the Thymosin alpha 1 PLGA microsphere emulsion that will prepare or emulsion 15～42 ℃ of temperature, stir under the condition that rotating speed is 20～3000 rev/mins.
Wherein, described temperature is 30.5-39 ℃, 300～2500 rev/mins of rotating speeds, and the method is called the intensification volatility process, can be preferred, stir as 1-24 hour.Further preferably, temperature is 33～39 ℃, and rotating speed is 500～1500 rev/mins, stirs 2～10 hours.Still more preferably, temperature is 34～36 ℃, and rotating speed is 600～800 rev/mins, stirs 5～6 hours.
Wherein, with the Thymosin alpha 1 PLGA microsphere emulsion for preparing or emulsion in vacuum-0.01～-15～40 ℃ of 0.1MP, temperature, the condition that rotating speed is 20～1000 rev/mins, the method are called the decompression volatility process.Further preferably, described vacuum-0.035～-26～35 ℃ of 0.096MP, temperature, the condition that rotating speed is 50～800 rev/mins, stir 2-18 hour.Still more preferably, described vacuum-0.08～-26～34 ℃ of 0.095MP, temperature, under the condition that rotating speed is 50～400 rev/mins, stirred 3～18 hours.Still more preferably, described vacuum is-0.08～-0.095MP, temperature is 26-34 ℃, rotating speed is under the condition of 120-400 rev/min, to stir 3～9 hours.
After completing above-mentioned steps, all filter according to a conventional method, washing, lyophilization.
The present invention controls the method for residue of organic solvent in thymic peptide alpha 1 microballoon, and efficiently controlled, process stabilizing, safe and reasonable, can effectively control the residue problem of the organic solvents such as dichloromethane, can keep the microsphere intact form simultaneously.
Below the present invention is described in further detail by the specific embodiment, but do not limit the present invention, those skilled in the art can make according to the present invention various changes and distortion, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
The specific embodiment
The organic solvent kind that remains in Thymosin alpha 1 PLGA microsphere of the present invention includes but not limited to: listed organic solvent in subordinate list 1, and chloroform, Ethyl formate, N, N-dimethyl formamide, oxirane, some emerging green solvents etc.
Organic solvent commonly used in subordinate list 1 medicine
1. usually contain 60% meta-xylene, 14% xylol, 9% o-Dimethylbenzene and 17% ethylbenzene.
The assay method of detection residue of organic solvent in thymic peptide alpha 1 microballoon of the present invention refers to the detection method of the conventional organic solvent residual that uses.Can measure with reference to gas chromatography (2005 editions appendix V E of Chinese Pharmacopoeia), as follows:
1. the selection of chromatographic column
1.1 the packed column immobile phase except as otherwise herein provided, between the close similar chromatographic column of polarity, in generation, use mutually.
(1) nonpolar chromatographic column fixative is the capillary column of 100% dimethyl polysiloxane.
(2) polarity chromatographic column fixative is the capillary column of Polyethylene Glycol (PEG-20M).
(3) Semi-polarity chromatographic column fixative is (35%) diphenyl-(65%) methyl polysiloxane, (50%) diphenyl-(50%) dimethyl polysiloxane, (35%) diphenyl-(65%) dimethyl arlydene polysiloxanes, (14%) cyanogen propyl group phenyl-(86%) dimethyl polysiloxane, the capillary column of (6%) cyanogen propyl group phenyl-(94%) dimethyl polysiloxane.
(4) low pole chromatographic column fixative is (5%) phenyl-(95%) methyl polysiloxane, the capillary column of (5%) diphenyl-(95%) dimethyl arlydene silicone copolymers.
1.2 the selection of packed column
Can select diameter is that the second diene benzene-EST type porous polymer beads of 0.25～0.18mm or other suitable fillers are as immobile phase.
2. the selection of assay method
Can adopt capillary column headspace sampling isothermal method
Chromatographic condition: column temperature is generally 40～100 ℃; Normal nitrogen buffer gas, flow velocity is 1.0～2.0ml per minute; Head space bottle heating-up temperature is 70～85 ℃, 30～60 minutes heat time heating times of head space bottle; Injector temperature is 200 ℃; As to adopt fid detector, temperature be 250 ℃.
Assay method: get reference substance solution and need testing solution, continuous sample introduction is no less than 2 times respectively, measures the peak area at peak to be measured.
In addition, can be with reference to " residual solvent algoscopy " in the Chinese Pharmacopoeia appendix about the selection of system suitability, head space condition, the selection of detector, interior target selection etc.Separately can carry out according to needs the checking of the methodology aspects such as specificity, detectability, quantitative limit, linearity, accuracy, ruggedness.
Below test adopts respectively direct lyophilization, room temperature volatility process, the present invention volatility process, the present invention volatility process that reduces pressure that heats up to carry out the control of organic solvent residual and the Determination of Residual Organic Solvents under more every kind of method.
1, investigate the preparation of sample
One (4 weeks of release time) of prescription
Ta1 1.0g
PLGA(0.15～0.25dl/g)（50/50） 36.0g
The organic solvent of selecting: dichloromethane
Preparation technology:, at normal temperatures with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 150ml dichloromethane, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 2000-2800 rev/min, water is injected oil phase, emulsifying 5-10 minute, obtain colostrum (O/W), and is standby;
To contain 3%PVA, the aqueous solution 800ml of 5%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 500-1000 rev/min, add colostrum, stirred 2 minutes, then add the 3%PVA that contains of uniform temp, the aqueous solution 200ml of 5%NaCl, obtain emulsion (W/O/W), and insulation is placed.
Prescription two
Ta1 1.0g
PLGA(0.70～0.90dl/g)（25/75） 32.0g
The organic solvent of selecting: pentane
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 100ml pentane, make it to dissolve fully, obtain oil phase, standby; Under 10-25 ℃, the mulser rotating speed is transferred to 2500-3200 rev/min, water is injected oil phase, emulsifying 5-10 minute, obtain colostrum (O/W), and is standby; To contain 2%PVA, the aqueous solution 1600ml of 3%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 500-1000 rev/min, add colostrum, stirred 2 minutes, then add the 2%PVA that contains of uniform temp, the aqueous solution 400ml of 3%NaCl, obtain emulsion (O/W/O), and insulation is placed.
Prescription three
Ta1 1.0g
PLGA(0.55～0.75dl/g)（75/25） 27.0g
The organic solvent of selecting: Ethyl formate
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 200ml Ethyl formate, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 3200-3800 rev/min, water is injected oil phase, emulsifying 5-10 minute, obtain colostrum (O/W), and is standby;
To contain 2%PVA, the aqueous solution 2500ml of 2%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, obtains the microsphere emulsion of W/O/W type, and insulation is placed.
2, adopt respectively direct lyophilization, room temperature volatility process, intensification volatility process, decompression volatility process further to prepare the emulsion that above-mentioned three formula preparations obtain, and detect the residual quantity of organic solvent in the finished product microsphere.
2.1 direct lyophilization
Get respectively emulsion or microsphere emulsion that three kinds of prescriptions make, stirred 3 hours under the condition of 500～1000 rev/mins, then be cooled to below 20 ℃; cross 100 mesh sieves, sucking filtration, drain, with distilled water wash 2 times; drain, lyophilization (adding GPF (General Protection False agent, proppant etc.), obtain microsphere.Sample thief, measure Determination of Residual Organic Solvents according to gas chromatography (2005 editions appendix V E of Chinese Pharmacopoeia).Continue the impact of drying time on Determination of Residual Organic Solvents for after investigating after sublimation drying, lyophilization the intensification temperature and heating up, choose respectively different time and temperature and investigate, investigate parameter and the results are shown in following table.
Subordinate list 2 Determination of Residual Organic Solvents measurement results
This experiment is for freeze-drying time, intensification temperature and investigate the drying time after heating up, extend to and far be more than common freeze-drying time (freeze-drying time is generally between 5～12 hours at freeze-drying time, minority reaches 24 hours) after 48 hours, can remove the organic solvent of a part, but the organic solvent that remains in microsphere still maintains a higher level.Have no significant effect Determination of Residual Organic Solvents the drying time after intensification temperature after lyophilizing and intensification.Above long lyophilizing, intensification, drying, can consume a large amount of energy and manpower and materials, makes production cost significantly increase energy resource consumption huge.
2.2.2 room temperature volatility process
Get respectively emulsion or microsphere emulsion that three kinds of prescriptions make, continue to stir under the condition of 500～1000 rev/mins under normal temperature and pressure conditions, be cooled to subsequently below 20 ℃ and cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization, obtain microsphere.Sample thief, measure Determination of Residual Organic Solvents according to gas chromatography (2005 editions appendix V E of Chinese Pharmacopoeia).For investigating the impact on Determination of Residual Organic Solvents of mixing speed, mixing time, choose respectively different speed and time and investigate, investigate parameter and the results are shown in following table.
Subordinate list 3 Determination of Residual Organic Solvents measurement results
| Lot number | Mixing speed (r/min) | Mixing time (h) | Determination of Residual Organic Solvents (%) |
| Prescription one | 500 | 5 | 3.1 |
| Prescription two | 500 | 10 | 2.8 |
| Prescription three | 500 | 48 | 2.6 |
| Prescription one | 750 | 5 | 2.4 |
| Prescription two | 750 | 10 | 2.0 |
| Prescription three | 750 | 36 | 2.1 |
| Prescription one | 1000 | 5 | 2.2 |
| Prescription two | 1000 | 10 | 1.8 |
| Prescription three | 1000 | 24 | 1.9 |
Take the room temperature volatility process,, by strengthening rotating speed, extending mixing time, can shoot the organic solvent of part, reduce the residual quantity of organic solvent in microsphere, but can't realize further reduction.
2.2.3 intensification volatility process
Get respectively emulsion or microsphere emulsion that three kinds of prescriptions make, stirred 3 hours under the condition of 500～1000 rev/mins, then be warming up to 30.5-42 ℃, continue to stir under the condition of 500～1000 rev/mins, then be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization, obtain microsphere.Sample thief, measure Determination of Residual Organic Solvents according to gas chromatography (2005 editions appendix V E of Chinese Pharmacopoeia).For investigating the impact on Determination of Residual Organic Solvents of mixing speed, rising temperature, mixing time, choose respectively different speed and time and investigate, investigate parameter and the results are shown in following table.
Subordinate list 4 Determination of Residual Organic Solvents measurement results
Result of the test shows, by the intensification in temperature range of the present invention and constant speed, stirs, and can further reduce the residual quantity of organic solvent on the basis of directly lyophilizing, room temperature volatility process by this law, and it is remained in a quite low limit.The microsphere that this law is made carries out scanning electron microscopic observation, and it is good that the form of microsphere keeps, without destroying.
2.2.4 decompression volatility process
Get respectively emulsion or microsphere emulsion that three kinds of prescriptions make, stirred 3 hours under the condition of 500～1000 rev/mins, then sample is transferred in eggplant type bottle, uses the rotary evaporator decompression, control temperature with the volatilization organic solvent, then be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization, obtain microsphere.Sample thief, measure Determination of Residual Organic Solvents according to gas chromatography (2005 editions appendix V E of Chinese Pharmacopoeia).For investigating the impact on Determination of Residual Organic Solvents of pressure, rotary speed, volatilization temperature, volatilization time, choose respectively different speed and time and investigate, investigate parameter and the results are shown in following table.
Subordinate list 5 Determination of Residual Organic Solvents measurement results
(vacuum=absolute atmosphere-one normal atmosphere, the vacuum ranges of upper table is :-0.005～-0.055MP)
Result of the test shows,, by controlling vacuum, temperature, mixing speed and mixing time in each parameter area of the present invention, can greatly reduce the residual quantity of organic solvent in microsphere, and it is remained in a quite low limit.
This test has adopted respectively direct lyophilization, stirring at normal temperature volatility process to remove the organic solvent in solution to the emulsion of three kinds of formula preparations, and finally measures the Determination of Residual Organic Solvents in microspheres product.Experimentation shows, the various influence factors dry by strengthening frozen and stirring at normal temperature is volatilized, can remove the part organic solvent in the Thymosin alpha 1 microsphere, but Determination of Residual Organic Solvents maintains all the time in a higher scope, can't further reduce, be unfavorable for directly for the patient.
The inventor is unexpected to be found, mixing speed by suitable control emulsion, the rising emulsion, to specified temp, can reduce the organic solvent that remains in microsphere within a certain period of time greatly, and other control indexs well to keep the form of microsphere and envelop rate, drug loading etc.; Namely pass through this special process when effectively controlling organic solvent residual, other character of microball preparation are unaffected.Under the guidance of this technique,, to specified temp, carry out agitation as appropriate by decompression, control emulsion, also can reach similar beneficial effect.
The specific embodiment of form by the following examples, foregoing of the present invention is described in further detail again, but this should be interpreted as that subject area of the present invention only limits to all technology that realizes based on content of the present invention of following example and all belongs to scope of the present invention.
Embodiment 1 the present invention controls the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.15～0.25dl/g)（50/50） 38.0g
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 150ml dichloromethane, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 2000-2800 rev/min, water is injected oil phase, emulsifying 5-10 minute, obtain colostrum (O/W), and is standby;
To contain 3%PVA, the aqueous solution 800ml of 5%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 500-1000 rev/min, adds colostrum, stirs 2 minutes, then adds the 3%PVA that contains of uniform temp, and the aqueous solution 200ml of 5%NaCl, obtain emulsion (O/W/O).Stirred 3 hours under the condition of 500～1000 rev/mins, sample is divided into two parts, adopts respectively the intensification volatility process: temperature (36 ℃), mixing speed (800-rev/min), stirring 6h and decompression volatility process: (0.095MPa), organic solvent is removed in temperature (31 ℃), rotating speed (250 rev/mins), stirring (8h) to vacuum.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 12 hours, obtain microsphere.The microsphere surrounding discharges fully, without the prominent phenomenon of releasing.
Embodiment 2 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.24～0.56dl/g)（50/50） 32.0g
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 100ml Ethyl formate, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 2500-3200 rev/min, water is injected oil phase, emulsifying 5-10 minute, obtain colostrum (O/W), and is standby;
To contain 2%PVA, the aqueous solution 1600ml of 3%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 500-1000 rev/min, adds colostrum, stirs 2 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 400ml of 3%NaCl, obtain emulsion (O/W/O).Stirred 3 hours under the condition of 500～1000 rev/mins, sample is divided into two parts, adopts respectively the intensification volatility process: temperature (37 ℃), mixing speed (900 rev/mins), stirring (7h) and decompression volatility process: (0.080MPa), temperature (26 ℃), rotating speed (400 rev/mins), stirring 9h remove organic solvent to vacuum.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 18 hours, obtain microsphere.The microsphere surrounding discharges fully, without the prominent phenomenon of releasing.
Embodiment 3 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.55～0.75dl/g)（75/25） 27g
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 200ml Ethyl formate, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 3200-3800 rev/min, water is injected oil phase, emulsifying 5-10 minute, obtain colostrum (O/W), and is standby;
To contain 2%PVA, the aqueous solution 2500ml of 2%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 400ml of 2%NaCl, obtain emulsion (W/O/W).Stirred 3 hours under the condition of 600～1200 rev/mins, sample is divided into two parts, adopt respectively the intensification volatility process: temperature (39 ℃), mixing speed (1500 rev/mins), stir 1h and decompression volatility process: vacuum (0.085MPa), temperature (32 ℃), rotating speed (80 rev/mins), stir 12h, remove organic solvent.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 12 hours, obtain microsphere.The microsphere surrounding discharges fully, without the prominent phenomenon of releasing.
Embodiment 4 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.55～0.75dl/g)（65/35） 38.5g
Mannitol 1.0g
Preparation technology
, with the Ta1 of recipe quantity and mannitol, join in the phosphate buffer of 35mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 400ml pentane, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 3800-4300 rev/min, water is injected oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
To contain 2%PVA, the aqueous solution 3500ml of 2%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 1500ml of 2%NaCl, obtain emulsion (W/O/W).Stirred 3 hours under the condition of 600～1200 rev/mins, sample is divided into two parts, adopt respectively the intensification volatility process: temperature (32 ℃), mixing speed (1800 rev/mins), stir 2h and decompression volatility process: vacuum (0.065MPa), temperature (35 ℃), rotating speed (800 rev/mins), stir 2h, remove organic solvent.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 24 hours, obtain microsphere.Three weeks of microsphere discharge fully, without the prominent phenomenon of releasing.
Embodiment 5 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.76～0.94dl/g)（25/75） 32.0g
PEG-6000 1.0g
Preparation technology
, with the Ta1 of recipe quantity and PEG-6000, join in the phosphate buffer of 40mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 280ml acetonitrile, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 4000-4500 rev/min, water is injected oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
To contain 2%PVA, the aqueous solution 4000ml of 2%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 2000ml of 2%NaCl, obtain emulsion (W/O/W).Stirred 3 hours under the condition of 600～1200 rev/mins, sample is divided into two parts, adopt respectively the intensification volatility process: temperature (38 ℃), mixing speed (1200 rev/mins), stir 3h and decompression volatility process: vacuum (0.092MPa), temperature (33 ℃), rotating speed (300 rev/mins), stir 6h, remove organic solvent.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 12 hours, obtain microsphere.Two weeks of microsphere discharge fully, without the prominent phenomenon of releasing.
Embodiment 6 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.76～0.94dl/g)（50/50） 11.0g
Preparation technology
, with the Ta1 of recipe quantity and mannitol, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 280ml dioxane, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 4000-4500 rev/min, water is injected oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
To contain 2%PVA, the aqueous solution 3000ml of 1.5%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 1000ml of 1.5%NaCl, obtain emulsion (W/O/W).Stirred 3 hours under the condition of 600～1200 rev/mins, sample is divided into two parts, adopt respectively the intensification volatility process: temperature (30.5 ℃), mixing speed (2000 rev/mins), time 1.5h and decompression volatility process: vacuum (0.035MPa), temperature (28 ℃), rotating speed (300 rev/mins), stir 4h, remove organic solvent.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 18 hours, obtain microsphere.Two weeks of microsphere discharge fully, without the prominent phenomenon of releasing.
Embodiment 7 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.76～0.94dl/g)（50/50） 22.0g
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 450ml dichloromethane, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 4000-4500 rev/min, water is injected oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
To contain 2%PVA, the aqueous solution 6000ml of 2%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 2000ml of 2%NaCl, obtain emulsion (W/O/W).Stirred 3 hours under the condition of 600～1200 rev/mins, sample is divided into two parts, adopts respectively the intensification volatility process: temperature (34 ℃), mixing speed (600 rev/mins), stirring (5h) and decompression volatility process: (0.090MPa), temperature (27 ℃), rotating speed (120 rev/mins), time (4h) are removed organic solvent to vacuum.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 12 hours, obtain microsphere.The microsphere surrounding discharges fully, without the prominent phenomenon of releasing.
Embodiment 8 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLGA(0.76～0.94dl/g)（50/50） 30.0g
Mannitol 1.0g
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 35mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLGA of recipe quantity at normal temperatures, join in the 500ml dichloromethane, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 4000-4500 rev/min, water is injected oil phase, emulsifying 5-10 minute, colostrum (O/W), standby;
To contain 2%PVA, the aqueous solution 6000ml of 2%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 2%PVA that contains of uniform temp, and the aqueous solution 2000ml of 2%NaCl, obtain emulsion (W/O/W).Stirred 3 hours under the condition of 600～1200 rev/mins, sample is divided into two parts, adopt respectively the intensification volatility process: temperature (31 ℃), mixing speed (350 rev/mins), stir 24h and decompression volatility process: vacuum (0.091MPa), temperature (34 ℃), rotating speed (400 rev/mins), stir 3h, remove organic solvent.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 12 hours, obtain microsphere.Three weeks of microsphere discharge fully, without the prominent phenomenon of releasing.
Embodiment 9 the present invention control the method for organic solvent residual in Thymosin alpha 1 PLGA microsphere
Ta1 1.0g
PLA(0.26～0..54dl/g) 59.0g
Preparation technology
, with the Ta1 of recipe quantity, join in the phosphate buffer of 30mlpH7.4 at normal temperatures, it is dissolved fully, obtain water, standby;
With the PLA of recipe quantity at normal temperatures, join in the 200ml dichloromethane, make it to dissolve fully, obtain oil phase, standby;
Under 10-25 ℃, the mulser rotating speed is transferred to 3500-4000 rev/min, water is injected oil phase, emulsifying 5-10 minute, obtain colostrum (O/W), and is standby;
To contain 3%PVA, the aqueous solution 3500ml of 5%NaCl is chilled to below 10 ℃, and the blender rotating speed is transferred to 600-1200 rev/min, adds colostrum, stirs 5 minutes, then adds the 3%PVA that contains of uniform temp, and the aqueous solution 1500ml of 5%NaCl, obtain emulsion (W/O/W).Stirred 3 hours under the condition of 600～1200 rev/mins, sample is divided into two parts, adopt respectively the intensification volatility process: temperature (33 ℃), mixing speed (500 rev/mins), stir 10h and decompression volatility process: vacuum (0.096MPa), temperature (35 ℃), rotating speed (50 rev/mins), stir 18h, remove organic solvent.Then all be cooled to below 20 ℃, cross 100 mesh sieves, sucking filtration, drain,, with distilled water wash 2 times, drain, lyophilization 24 hours, obtain microsphere.12 weeks of microsphere discharge fully, without the prominent phenomenon of releasing.
The final products Determination of Residual Organic Solvents of each embodiment, envelop rate, particle size data are as follows:
By above-described embodiment, as can be known, wherein, the condition of intensification volatility process is: temperature 30.5-42 ℃, under the condition that rotating speed is 350～2000 rev/mins, stir 1-24 hour.The condition of decompression volatility process is: 26～35 ℃ of vacuum-0.005--0.096MP, temperature, and the condition that rotating speed is 50～800 rev/mins, mixing time is: 2-18 hour.In process conditions of the present invention, all the residual quantity of organic solvent can be controlled lower than 0.06%, and do not affected envelop rate and the particle diameter of Thymosin alpha 1 PLGA microsphere, microsphere release is complete in addition, without the prominent phenomenon of releasing.
Claims (3)
1. method of controlling organic solvent residual in Thymosin alpha 1 PLGA microsphere is characterized in that:
The Thymosin alpha 1 PLGA microsphere emulsion that (1) will prepare or emulsion stirred 3 hours under the condition of 500～1200 rev/mins of normal temperature and pressures;
(2) in vacuum-0.035～-26～35 ℃ of 0.096MP, temperature, under the condition that rotating speed is 50～800 rev/mins, stir 2-18 hour.
2. method according to claim 1, is characterized in that, vacuum-0.08 in step (2)～-26～34 ℃ of 0.095MP, temperature, 50～400 rev/mins of rotating speeds, stirred 3～18 hours.
3. method according to claim 2, is characterized in that, in step (2) vacuum be-0.08～-0.095MP, temperature is 26-34 ℃, rotating speed is 120-400 rev/min, stirs 3～9 hours.
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