CN107556327A - A kind of method for isolating and purifying tacrolimus - Google Patents
A kind of method for isolating and purifying tacrolimus Download PDFInfo
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- CN107556327A CN107556327A CN201711049488.7A CN201711049488A CN107556327A CN 107556327 A CN107556327 A CN 107556327A CN 201711049488 A CN201711049488 A CN 201711049488A CN 107556327 A CN107556327 A CN 107556327A
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- tacrolimus
- isolating
- purifying
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- eluent
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Abstract
The invention provides a kind of method for isolating and purifying tacrolimus, the present invention use two sets of medium pressure chromatography chromatographic equipments, using low chromatographic purity tacrolimus fermentation extract solution as raw material, by impurity such as first thin layer chromatography post Polysaccharide removing, protein；Extraction solvent then is removed with rotary evaporation technique, dissolves again and carries out second thin layer chromatography post separation and go out low concentration and the eluent containing tacrolimus, eluent merges through excessive batch, high-purity is obtained after being concentrated using NF membrane and in the tacrolimus of solid-state.Pass through a kind of disclosed method for isolating and purifying tacrolimus, it can be achieved directly to carry out purification process from the zymotic fluid containing tacrolimus, and the purity of tacrolimus is considerably improved, and improve indirectly as the drug effect of the immune formulation made by tacrolimus.
Description
Technical field
The present invention relates to biopharmaceutical technology, more particularly to a kind of method for isolating and purifying tacrolimus.
Background technology
Tacrolimus also known as FK-506 (Tacrolimus), it is from building ripple strepto- by Japanese Teng Ze drugmakers in nineteen eighty-two
A kind of macrolide immunosuppressants extracted in bacterium metabolite.This product is main to the selective inhibitory action of T cell
If discharging IL-2, IL-3, IFN-γ by suppressing TH cells, and suppress IL-2R expression and to play its powerful immune
Inhibitory action.Suitable for the rejection after preventing and treating organ transplant, its clinical efficacy is 100 times of ring born of the same parents element.
At present, tacrolimus is mainly prepared by fermentation method, therefore can produce many isomers during the fermentation
Or the like, the technical scheme isolated and purified using traditional reversed-phase resin, modified silica-gel finally purifies what is obtained
The purity of tacrolimus is unsatisfactory, is typically only capable to reach 98% or so.All polyisocyanates are still had in tacrolimus after purification
The impurity such as structure body, so as to be affected to the drug effect as the immune formulation made by tacrolimus, and may be to patient
Cause larger side effect.
In view of this, it is necessary to which the technical scheme that tacrolimus of the prior art is separated and purified is changed
Enter, to solve the above problems.
The content of the invention
It is an object of the invention to disclose a kind of method for isolating and purifying tacrolimus, he is prepared to fermentation method to realize
Purification process is directly carried out in the zymotic fluid containing tacrolimus that Ke Mosi is formed, to prepare the tacrolimus of high-purity,
To improve as the drug effect of the immune formulation made by tacrolimus.
To achieve the above object, the invention provides a kind of method for isolating and purifying tacrolimus, comprise the following steps：
(1) low chromatographic purity tacrolimus fermentation extract solution is prepared, after being filtered using the organic filter membrane of 0.45 micron of micropore,
It is stand-by；
(2) chromatographic silica gel is used to enter as filler using acetone and/or ethanol in first set medium pressure chromatography chromatographic equipment
Row cleaning and balance；
(3) low chromatographic purity tacrolimus fermentation extract solution is chromatographed in first set medium pressure chromatography chromatographic equipment, used
Organic solvent is eluted, and is collected eluent, eluent rotary evaporation, 0.45 micron is reused after being dissolved with organic solvent
The organic filter membrane of micropore filters；
(4) reverse silica gel is used in second set of medium pressure chromatography chromatographic equipment as filler, it is stand-by；
(5) reverse silica gel is cleaned using the organic solvent at least containing acetone, and gained sample liquid in step (3) is configured to fit
The solution of suitable loading, eluted by multistage after loading, obtain eluent；
(6) by more batches of merging of gained eluent in step (5), concentrated using NF membrane, and carry out decolorization；
(7) gained concentrate in step (6) is crystallized, and dried.
As a further improvement on the present invention, the chromatographic purity of tacrolimus fermentation extract solution is in the step (1)
60%.
As a further improvement on the present invention, the particle diameter of the chromatographic silica gel in the step (2) is 300~400 mesh, pressure
Condition is 60psi~90psi.
As a further improvement on the present invention, the evaporating temperature of the rotary evaporation in the step (3) is 35 DEG C of temperature, is turned
Speed is 70rpm, and vacuum is -0.09MPa~-0.1MPa.
As a further improvement on the present invention, the molecular weight of NF membrane selected in the step (6) is 200, concentration
Condition is：20 DEG C~60 DEG C of temperature, pressure 0.8MPa~1.2MPa.
As a further improvement on the present invention, in the step (7), the concentrate before crystallization should meet following condition：
Concentration is 75g/L~80g/L, PH9.30~9.60.
As a further improvement on the present invention, the crystallization in the step (7) is specially：Use pure steam heating stepses
(6) concentrate of gained is immediately filtered to 35 DEG C~55 DEG C using 0.45 micron of the organic filter membrane of micropore in, and with 0 DEG C
~70 DEG C of acetone elution, to be crystallized.
As a further improvement on the present invention, the drying in the step (7) is specially：Tacrolimus crystal after crystallization
Be placed in vacuum drying oven and be dried, the drying temperature in the vacuum drying oven is set as 30 DEG C~40 DEG C, vacuum drying oven it is true
Reciprocal of duty cycle is -0.098MPa~-0.102MPa.
Compared with prior art, the beneficial effects of the invention are as follows：Him is isolated and purified gram by disclosed one kind
The method do not taken charge of, the directly progress purification process, and considerably improve him gram from the zymotic fluid containing tacrolimus can be achieved
The purity do not taken charge of, improve indirectly as the drug effect of the immune formulation made by tacrolimus.
Brief description of the drawings
Fig. 1 for embodiment 1 final obtained tacrolimus HPLC collection of illustrative plates；
Fig. 2 for embodiment 2 final obtained tacrolimus HPLC collection of illustrative plates.
Embodiment
The present invention is described in detail for shown each embodiment below in conjunction with the accompanying drawings, but it should explanation, these
Embodiment is not limitation of the present invention, those of ordinary skill in the art according to these embodiment institute work energy, method,
Or equivalent transformation or replacement in structure, belong within protection scope of the present invention.
Embodiment 1：
A kind of method for isolating and purifying tacrolimus shown by the present embodiment, comprises the following steps：
Step (1)：Low chromatographic purity tacrolimus fermentation extract solution 250g is prepared, the micropore using 0.45 micron has machine filter
It is stand-by after film filters.Wherein, the chromatographic purity of tacrolimus fermentation extract solution is 60% or so.
Step (2)：Chromatographic silica gel is used to be carried out as filler using ethanol clear in first set medium pressure chromatography chromatographic equipment
Wash and balance.The particle diameter of chromatographic silica gel in the step (2) is 300 mesh, pressure condition 60psi.In the present embodiment, lead to
First set medium pressure chromatography chromatographic equipment is crossed, effectively removes polysaccharide, egg in low chromatographic purity tacrolimus fermentation extract solution
The big molecular impurities such as white matter, operation is further purified in favor of the later stage.
Step (3)：Low chromatographic purity tacrolimus fermentation extract solution is chromatographed in first set medium pressure chromatography chromatographic equipment,
Using organic solvent (such as：By acetone and ethanol according to 1:The mixed solution that 2 mol ratio is formed) eluted, collection is washed
De- liquid, eluent rotary evaporation, with organic solvent (such as：Acetone) 0.45 micron of the organic filter membrane of micropore is reused after dissolving
Filter.Wherein, the evaporating temperature of the rotary evaporation in step (3) is 35 DEG C of temperature, rotating speed 70rpm, vacuum for-
0.09MPaMPa。
Step (4)：Reverse silica gel is used in second set of medium pressure chromatography chromatographic equipment as filler, it is stand-by.
Step (5)：Reverse silica gel is cleaned using the organic solvent at least containing acetone, and gained sample liquid in step (3) is matched somebody with somebody
The solution of suitable loading is set to, is eluted after loading by multistage, obtains eluent.
Step (6)：By more batches of merging of gained eluent in step (5), concentrated using NF membrane, and carry out decolorization.
Specifically, the molecular weight of NF membrane selected in step (6) is 200, concentration condition is：20 DEG C of temperature, pressure 0.8MPa.
In the present embodiment, merging the number of eluent can be once, or twice or three times, be less than with concentrate electrical conductivity or
It is criterion equal to 280 μ s/cm.
Step (7)：Gained concentrate in step (6) is crystallized, and dried, so that the tacrolimus powder of high-purity is made
End.
Specifically, in step (7), the concentrate before crystallization should meet following condition：Concentration is 75g/L, PH9.30.Step
Suddenly the crystallization in (7) is specially：Using the concentrate of gained in pure steam heating stepses (6) to 35 DEG C, immediately use
0.45 micron of the organic filter membrane of micropore filters, and is eluted with 0 DEG C of acetone, to be crystallized.Drying in step (7) is specific
For：Tacrolimus crystal after crystallization, which is placed in vacuum drying oven, to be dried, and the drying temperature in the vacuum drying oven is set as
30 DEG C, the vacuum of vacuum drying oven is -0.098MPa.
Join shown in Fig. 1, the high-purity tacrolimus powder that the present embodiment is finally prepared uses high performance liquid chromatography
Detection, the purity for measuring tacrolimus are 99%, total recovery 35%.Wherein, high-efficiency liquid chromatography method for detecting is marked with reference to EP8.0
Standard, the filler as stationary phase are Agela Venusil XBPC18 (A), particle diameter：5 μm, post specification：4.6 × 250mm or performance
Suitable post, flow velocity 1.0ml/min, 60 DEG C, Detection wavelength 232nm of column temperature, the μ l of sample size 20.Mobile phase is pH6.5 phosphate
Cushioning liquid, acetonitrile and water are with 5:38:57 ratios (mol ratio) mix, and the ratio can suitably adjust the guarantor for making tacrolimus component
It is 23~27min to stay the time, in order to which tacrolimus is separated by more effectively elution.Meanwhile in the present embodiment with embodiment 1
The technical scheme of same section, join described in embodiment 1, will not be repeated here.
Embodiment 2：
A kind of method for isolating and purifying tacrolimus shown by the present embodiment, comprises the following steps：
Step (1)：Low chromatographic purity tacrolimus fermentation extract solution is prepared, is taken out using the organic filter membrane of 0.45 micron of micropore
It is stand-by after filter.Wherein, the chromatographic purity of tacrolimus fermentation extract solution is 60% or so.
Step (2)：Chromatographic silica gel is used to be carried out as filler using acetone clear in first set medium pressure chromatography chromatographic equipment
Wash and balance.The particle diameter of chromatographic silica gel in step (2) is 400 mesh, pressure condition 90psi.
Step (3)：Low chromatographic purity tacrolimus fermentation extract solution is chromatographed in first set medium pressure chromatography chromatographic equipment,
Using organic solvent (such as：Ethanol) eluted, collect eluent, eluent rotary evaporation, with organic solvent (such as：Third
Ketone or ethanol) the organic filter membrane of micropore that reuses 0.45 micron after dissolving filters.The evaporation of rotary evaporation in step (3)
Temperature is 35 DEG C of temperature, and rotating speed 70rpm, vacuum is -0.1MPa.
Step (4)：Reverse silica gel is used in second set of medium pressure chromatography chromatographic equipment as filler, it is stand-by.
Step (5)：Reverse silica gel is cleaned using the organic solvent at least containing acetone, and gained sample liquid in step (3) is matched somebody with somebody
The solution of suitable loading is set to, is eluted after loading by multistage, obtains eluent.
Step (6)：By more batches of merging of gained eluent in step (5), concentrated using NF membrane, and carry out decolorization.
Specifically, the molecular weight of NF membrane selected in step (6) is 200, concentration condition is：Temperature 60 C, pressure 1.2MPa.
In the present embodiment, merging the number of eluent can be once, or twice or three times, be less than with concentrate electrical conductivity or
It is criterion equal to 280 μ s/cm.
Step (7)：Gained concentrate in step (6) is crystallized, and dried, so that the tacrolimus powder of high-purity is made
End.
Specifically, in step (7), the concentrate before crystallization should meet following condition：Concentration is 80g/L, PH9.60.Step
Suddenly the crystallization in (7) is specially：Using the concentrate of gained in pure steam heating stepses (6) to 55 DEG C, immediately use
0.45 micron of the organic filter membrane of micropore filters, and is eluted with 70 DEG C of acetone, to be crystallized.Drying in step (7) is specific
For：Tacrolimus crystal after crystallization, which is placed in vacuum drying oven, to be dried, and the drying temperature in the vacuum drying oven is set as
40 DEG C, the vacuum of vacuum drying oven is -0.102MPa.
Join shown in Fig. 2, the high-purity tacrolimus powder that the present embodiment is finally prepared uses high performance liquid chromatography
Detection, the purity for measuring tacrolimus are 99%, total recovery 36%.The experimental condition ginseng embodiment 1 of high performance liquid chromatography detection
It is described, it will not be repeated here.
Those listed above is a series of to be described in detail only for feasibility embodiment of the invention specifically
Bright, they simultaneously are not used to limit the scope of the invention, all equivalent implementations made without departing from skill spirit of the present invention
Or change should be included in the scope of the protection.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Any reference in claim should not be considered as to the involved claim of limitation.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (8)
- A kind of 1. method for isolating and purifying tacrolimus, it is characterised in that comprise the following steps：(1) low chromatographic purity tacrolimus fermentation extract solution is prepared, after being filtered using the organic filter membrane of 0.45 micron of micropore, is treated With；(2) chromatographic silica gel is used to be carried out as filler using acetone and/or ethanol clear in first set medium pressure chromatography chromatographic equipment Wash and balance；(3) low chromatographic purity tacrolimus fermentation extract solution is chromatographed in first set medium pressure chromatography chromatographic equipment, use is organic Solvent is eluted, collection eluent, eluent rotary evaporation, and 0.45 micron of micropore is reused after being dissolved with organic solvent Organic filter membrane filters；(4) reverse silica gel is used in second set of medium pressure chromatography chromatographic equipment as filler, it is stand-by；(5) reverse silica gel is cleaned using the organic solvent at least containing acetone, and gained sample liquid in step (3) is configured on suitable The solution of sample, eluted by multistage after loading, obtain eluent；(6) by more batches of merging of gained eluent in step (5), concentrated using NF membrane, and carry out decolorization；(7) gained concentrate in step (6) is crystallized, and dried.
- 2. the method according to claim 1 for isolating and purifying tacrolimus, it is characterised in that in the step (1) he gram The chromatographic purity of department's fermentation extract solution is not 60%.
- 3. the method according to claim 1 for isolating and purifying tacrolimus, it is characterised in that the layer in the step (2) The particle diameter for analysing silica gel is 300~400 mesh, and pressure condition is 60psi~90psi.
- 4. the method according to claim 1 for isolating and purifying tacrolimus, it is characterised in that the rotation in the step (3) The evaporating temperature for turning evaporation is 35 DEG C of temperature, and rotating speed 70rpm, vacuum is -0.09MPa~-0.1MPa.
- 5. the method according to claim 1 for isolating and purifying tacrolimus, it is characterised in that selected in the step (6) The molecular weight of NF membrane is 200, and concentration condition is：20 DEG C~60 DEG C of temperature, pressure 0.8MPa~1.2MPa.
- 6. the method according to claim 1 for isolating and purifying tacrolimus, it is characterised in that in the step (7), crystallization Preceding concentrate should meet following condition：Concentration is 75g/L~80g/L, PH9.30~9.60.
- 7. the method according to claim 1 for isolating and purifying tacrolimus, it is characterised in that the knot in the step (7) Crystalline substance is specially：Using the concentrate of gained in pure steam heating stepses (6) to 35 DEG C~55 DEG C, immediately using 0.45 micron The organic filter membrane of micropore filter, and with 0 DEG C~70 DEG C of acetone elution, to be crystallized.
- 8. the method according to claim 1 for isolating and purifying tacrolimus, it is characterised in that the baking in the step (7) It is dry to be specially：Tacrolimus crystal after crystallization, which is placed in vacuum drying oven, to be dried, the drying temperature in the vacuum drying oven It is set as 30 DEG C~40 DEG C, the vacuum of vacuum drying oven is -0.098MPa~-0.102MPa.
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| CN201711049488.7A CN107556327A (en) | 2017-10-31 | 2017-10-31 | A kind of method for isolating and purifying tacrolimus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111450574A (en) * | 2019-01-22 | 2020-07-28 | 福州奥尼多生物科技有限公司 | Chromatographic column for purifying tacrolimus and purification method of tacrolimus |
| WO2024164432A1 (en) * | 2023-02-06 | 2024-08-15 | 华北制药股份有限公司 | Tacrolimus sustained-release preparation and preparation method therefor |
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| WO2005019226A1 (en) * | 2003-08-26 | 2005-03-03 | Biocon Limited | A process for the recovery of substantially pure tricyclic macrolide |
| CN101048415A (en) * | 2004-11-03 | 2007-10-03 | 安蒂比奥蒂科斯有限公司 | Process for the purification of tacrolimus |
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2017
- 2017-10-31 CN CN201711049488.7A patent/CN107556327A/en active Pending
Patent Citations (3)
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| WO2005019226A1 (en) * | 2003-08-26 | 2005-03-03 | Biocon Limited | A process for the recovery of substantially pure tricyclic macrolide |
| CN101048415A (en) * | 2004-11-03 | 2007-10-03 | 安蒂比奥蒂科斯有限公司 | Process for the purification of tacrolimus |
| CN107056814A (en) * | 2017-02-10 | 2017-08-18 | 广州市微生物研究所 | A kind of preparation method of the crude tacrolimus of low stain |
Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111450574A (en) * | 2019-01-22 | 2020-07-28 | 福州奥尼多生物科技有限公司 | Chromatographic column for purifying tacrolimus and purification method of tacrolimus |
| CN111450574B (en) * | 2019-01-22 | 2022-01-21 | 福州奥尼多生物科技有限公司 | Chromatographic column for purifying tacrolimus and purification method of tacrolimus |
| WO2024164432A1 (en) * | 2023-02-06 | 2024-08-15 | 华北制药股份有限公司 | Tacrolimus sustained-release preparation and preparation method therefor |
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Application publication date: 20180109 |