CN108864257A - High purity cyclosporin A and preparation method thereof - Google Patents
High purity cyclosporin A and preparation method thereof Download PDFInfo
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- CN108864257A CN108864257A CN201810684846.XA CN201810684846A CN108864257A CN 108864257 A CN108864257 A CN 108864257A CN 201810684846 A CN201810684846 A CN 201810684846A CN 108864257 A CN108864257 A CN 108864257A
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- cyclosporin
- 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
- C07K7/64—Cyclic peptides containing only normal peptide links
- C07K7/645—Cyclosporins; Related peptides
Abstract
The invention discloses a kind of new high purity cyclosporin As and preparation method thereof.By cyclosporin A filtering fermentation liquor, bacteria residue alcohol steep is concentrated, and concentrate adds base extraction, can remove a large amount of impurity, while being conducive to extracting and demixing.Extract liquor is decolourized, and crystallization obtains the crude product of 90% or more purity.The cyclosporin A fine work of purity 99.5% can be obtained through column chromatography, crystallization purifying for crude product.Method provided by the invention is easy to operate, high income, and solvent consumption is few, with short production cycle, at low cost, is suitable for industrialized production, and cyclosporin A purity is high obtained improves the quality and safety of drug.
Description
Technical field
The present invention relates to drug technical field of purification, and in particular to a kind of preparation method and its use of cyclosporin A crude product
In the method for efficiently preparing high purity cyclosporin A.
Background technique
Cyclosporin A is a kind of ring type polypeptide compound containing 11 amino acid, is a kind of very strong immune suppression of effect
Preparation, the anti-repelling treatment after being widely used in the solid organ transplantations such as kidney, liver, heart and lung.It can be also used for controlling
Adult atopic dermatitis is treated, and for treating some autoimmune diseases.
Currently, the method that cyclosporin A mainly passes through microbial fermentation obtains.In the fermentation liquid that fermentation is completed, in addition to containing
Have other than cyclosporin A, also containing other several cyclosporins and medium components, various bacterial metabolisms such as B, C, D, G, H
Other impurity such as object.The prior art is first to be extracted with organic solvent to the dominating process route that cyclosporin A purifies, and is then passed through
Different types of column chromatography is isolated and purified in conjunction with crystallization.
US5656459 is disclosed after the culture medium static fermentation of the non-active ingredient of certain content range composition, fermentation
Liquid obtains a residue with methanol extraction, and secondary retained object is obtained by extraction through ethyl acetate after a residue decoloration, secondary
Residue is purified by silica gel column chromatography, and affords residue three times with n-hexane/chloroform/methanol, finally pure with resin column again
Change, methanol elution obtains cyclosporin A.This method obtains cyclosporin A crude product through conventional extraction twice, and crude product is miscellaneous before column chromatographs
Matter type and content are more, need column chromatographic purifying twice, and do eluant, eluent, production cost using the chloroform and methanol being more toxic
Height, environment are unfriendly, are not suitable for industrialized production.
WO20010064935, which is disclosed, will be added alum stirring 3 hours, mistake after fermentation liquid methanol extraction, in concentrate
Filter, filter cake are dissolved with hexamethylene, the cyclosporin A crude product that purity is 59.8% are obtained after concentration, crude product is again through gel filtration, column
Chromatography and crystallization purifying obtain cyclosporin A product.
CN200510108097.9, which is disclosed, directly adds acetone, filtering into fermentation liquid, is concentrated under reduced pressure, then use ethyl acetate
Extraction, organic phase use acetone/water/n-hexane three phase extraction twice after being concentrated to dryness, then are crystallized 3 days with acetone/water in -5 DEG C, silicon
Glue 60 carries out column chromatographic purifying, and active carbon decoloring is finally recrystallized to give the production that purity is greater than 98.5% under the conditions of -15 DEG C
Product, total recovery 55.1%-64.9%.The technique need to be extracted repeatedly, wherein be difficult to be layered when acetone/water/n-hexane three phase extraction,
60 filler of silica gel used is expensive, and crystallization needs 3 days for the first time, and time-consuming for entire technique.
CN200910253905.9 discloses the separation method that a kind of large pore resin absorption column and silica gel column chromatography combine.
Into fermentation liquid plus NaOH solution adjusts pH to neutrality, and organic solvent is added and extracts cyclosporin A.Successively use macroporous absorbent resin
Chromatography and silica gel column chromatography purifying, collect the cyclosporin A eluent of purity qualification, and it is big to obtain purity for last crystallization for purifying
In 98.5% finished product, total recovery 65.3-70.1%.This method needs column chromatography twice, and solvent usage is big, high production cost, no
It is suitble to industrialized production.
Separately there are other tens of documents to disclose the isolation and purification method of cyclosporin A.As patent US597638 is disclosed
A method of cyclosporin A being obtained by silica gel column chromatography using high-pressure carbon dioxide as flowing carrier；《Chinese antibiotic
Magazine, 2008,5,276-280》It is isolated and purified using macroporous absorbent resin combination high speed adverse current chromatogram；US5382655 is disclosed
A kind of silica gel column chromatography method etc. for using chloroform, methylene chloride and ethyl alcohol to isolate and purify cyclosporin A as mobile phase.
It, need to be through silica gel column layer after isolation and purification method in the prior art is summarized it is found that fermentation liquid extracts with organic solvent
Other chromatographies of analysis joint or crystallization purifying, or could obtain using the chromatography of special installation and compare the cyclosporin of high-purity
A.This is because containing more impurity, as disclosed in WO20010064935, crude product in the crude product that separation and Extraction early period obtains
Purity is 59.8%.The prior art generally using after fermentation liquid is extracted, extracted, improves crude product by pre- chromatographic purifying
Purity, but the crude product purity obtained is unsatisfactory, and the purity of commercially available cyclosporin A crude product is about 65%, and column chromatographs at this time
Applied sample amount is very low, high production cost.Therefore, it in order to efficiently obtain the cyclosporin A of high-purity, develops new, easily operated
Early period, purifying process, reduced impurity content, is conducive to subsequent acquisition high purity product, has very important significance.
Summary of the invention
Impurity is more when the object of the invention is in order to overcome purifies and separates cyclosporin A fermentation liquid, and separating difficulty is big, existing
There is the deficiency that purification process is complicated, time-consuming, crude intermediate quality is low, column chromatography cyclosporin A applied sample amount is low.One kind is provided
The cyclosporin A crude product of higher degree is obtained easy to operately and efficiently prepares the cyclosporin A of high-purity using the crude product
Method.Solve that prior art complexity, complex steps, time-consuming for purification process, silica gel and eluting solvent dosage are big, environment is not friendly
Well, it is unfavorable for the problem of industrialized production.
In order to solve the above technical problem, the present invention provides following technical schemes：
Present invention firstly provides a kind of preparation methods of cyclosporin A crude product, it is characterised in that：Include the following steps：
(1), by cyclosporin filtering fermentation liquor, bacteria residue is concentrated after being parsed with ethyl alcohol, drives away whole ethyl alcohol or part ethyl alcohol,
Obtain concentrate；
(2), concentrate adds base extraction to obtain treatment fluid；
(3), treatment fluid is extracted with organic solvent, and decoloration crystallizes to obtain cyclosporin A crude product after concentration.
Wherein, gained crude product purity is 90% or more.
Base extraction is added in extracting solution, the saponification of generation can remove the impurity such as a large amount of greases, while also be conducive to extraction
Take layering.The cyclosporin A crude product obtained after crystallization the extracting solution intermediate more commonly used in the prior art as sample solution is more
Stablize, is conducive to save.
In a preferred embodiment, lye described in step (2) is selected from NaOH solution, KOH solution or Na2CO3It is molten
Liquid, preferably NaOH solution；
In a preferred embodiment, concentration of lye described in step (2) is 0.5-1.5N, preferably 1N；
In a preferred embodiment, described in step (2) plus base extraction is plus lye is to pH=9.5~11.5,
And in 50~60 DEG C stirred in water bath 2 hours, preferably 60 DEG C.
In a preferred embodiment, organic solvent described in step (3) is ethyl acetate, ethyl acetate and treatment fluid
Volume ratio be 3-4: 1.
In a preferred embodiment, decoloration described in step (3) is that the work of extract liquor volume 2-4% (W/V) is added
Property charcoal, stir 2 hours, be filtered, washed to obtain destainer.
In a preferred embodiment, the method for crystallization described in step (3) is to be added third in the residue after concentration
The w/v of ketone, residue and acetone is 0.8-1.5: 1, is placed at -5 DEG C to -18 DEG C crystallization 20 hours or more.
The cyclosporin A of 99.5% or more purity can be made using cyclosporin A crude product obtained by the present invention.General fermentation liquid
By extraction, repeatedly extraction or a macroreticular resin are isolated and purified, and gained cyclosporin A intermediate is through column chromatographic purifying
When, upper column capacity is the 20-45mg total hundred million of every gram of chromatography media, and it is 60% or so that column, which chromatographs yield, and with column chromatographic purifying sheet
Resulting cyclosporin A crude product is invented, for upper column capacity up to the 50-75mg of every gram of chromatography media total hundred million, it is reachable that column chromatographs yield
80% or more.It collects cyclosporin A component and is greater than 98.5% chromatography fraction, be concentrated, crystallization is met the requirements in high yield
High purity cyclosporin A fine work.
Compared with prior art, the unexpected technical effect that the present invention obtains is as follows：
1, the present invention greatly improves the purity of cyclosporin A crude product, operates by extracting solution through simple post-processing purifying
Simply, equipment and solvent are saved, it is environmental-friendly, it is at low cost, it is very suitable to industrialized production, and the product obtained is easily stored；
2, the cyclosporin A finished product of high-purity can be obtained in high yield using cyclosporin A crude product provided by the invention, it is pure
Degree improves the quality and safety of drug up to 99.5% or more.
Detailed description of the invention
Fig. 1 is the crystal scanning figure of 1 gained cyclosporin A crude product of the embodiment of the present invention.
Fig. 2 is the HPLC spectrogram of 1 gained cyclosporin A crude product of the embodiment of the present invention.
Specific embodiment
In order to better understand the content of the present invention, technical solution of the present invention is done into one combined with specific embodiments below
The explanation of step, but specific embodiment is not meant to there are any restrictions to the present invention.
The instrument that the present invention uses is as follows：
Sem analysis is carried out to crystal form using Hitachi, Japan S-4800 type scanning electron microscope, operation voltage is 15.0kV,
Electric current 10 μ A, operating distance 10.3mm.Sample carries out surface metal spraying processing, vacuum degree under vacuum before being observed
10Pa, metal spraying time 60s.
High performance liquid chromatography (HPLC) spectral data is picked up from high performance liquid chromatograph：Agilent 1260；
Chromatographic column：C18 column (4.6 × 250mm, 5 μm)；
Mobile phase：Acetonitrile: water: phosphoric acid: methyl tertiary butyl ether(MTBE)=490: 460: 1: 50
Ultraviolet detection wavelength：210nm；
Column temperature：80℃；
Flow velocity：1.5mL/min；Sample volume：20uL.
Solvent for use of the present invention is commercially available to be bought.
The preparation of 1 cyclosporin A crude product of embodiment
5L cyclosporin A filtering fermentation liquor is obtained into bacteria residue, 65 DEG C drying 4 hours, 2.5L ethyl alcohol is added in the dry bacteria residue of gained
Solution stirs 4 hours, filtering, and 2L ethyl alcohol is added in filter residue and extracts again, and operation is same to be extracted for the first time.Filtering, merging are soaked twice
Extract is concentrated under reduced pressure at 60 DEG C, obtains concentrate.
Into gained concentrate, the NaOH solution of addition 1N is about to stir 2 hours under 10,60 DEG C of water-baths to pH, is added
The extraction of 500mL ethyl acetate isolates organic phase, 10g activity carbon decoloring is added in organic phase, stirs 1 hour, filtering, filtrate
It is concentrated under reduced pressure, 60mL acetone is added in residue, stirs evenly, 0.5g cyclosporin A crystal seed is added, is placed under -10 DEG C of environment
Crystallization 20 hours is filtered, dry, obtains cyclosporin A crude product, total recovery 81.3%, purity 92.0%.Crude crystalline scanning
Figure is shown in that Fig. 1, HPLC spectrogram are shown in Fig. 2.
The preparation of 2 cyclosporin A crude product of embodiment
5L cyclosporin A fermentation liquid is taken, bacteria residue is filtered to obtain, 65 DEG C obtain dry bacteria residue in drying 4 hours.The dry bacteria residue of gained is added
It into 2.5L ethanol solution, stirs 4 hours, filtering, 2L ethyl alcohol is added in filter residue and extracts again, operation is same to be extracted for the first time.It crosses
Filter merges leaching liquor twice, is concentrated under reduced pressure at 60 DEG C, obtains concentrate.
Into gained concentrate, the KOH solution of addition 1N is about 9.5 to pH, is placed in 60 DEG C of stirred in water bath 2 hours, is added
The extraction of 500mL ethyl acetate isolates organic phase, 10g activity carbon decoloring is added in organic phase, stirs 1 hour, filters, uses
100mL ethyl acetate elutes active carbon, and filtrate decompression is concentrated, and 60mL acetone is added in residue, stirs evenly, and 0.5g ring is added
Spore rhzomorph A crystal seed places crystallization 20 hours under -10 DEG C of environment, filters, dry, obtains cyclosporin A crude product, total recovery 80.1%,
Purity 90.8%.
The preparation of 3 cyclosporin A crude product of embodiment
5L cyclosporin A fermentation liquid is taken, bacteria residue is filtered to obtain, 65 DEG C obtain dry bacteria residue in drying 4 hours.2.5L is added in dry bacteria residue
Ethyl alcohol stirs 4 hours, filtering, and 2L ethyl alcohol is added in filter residue and extracts again, and operation is same to be extracted for the first time.Filtering, merging are soaked twice
Extract is concentrated under reduced pressure at 60 DEG C, obtains concentrate.
The Na of 1N is added into gained concentrate2CO3Solution is about 11.5 to pH, is placed in 60 DEG C of stirred in water bath 2 hours,
The extraction of 500mL ethyl acetate is added, isolates organic phase, 10g activity carbon decoloring is added in organic phase, stirs 1 hour, filtering,
Active carbon is eluted with 100mL ethyl acetate, filtrate decompression is concentrated, and 55mL acetone is added in residue, stirs evenly, and 0.5g is added
Cyclosporin A crystal seed places crystallization 20 hours under -10 DEG C of environment, filters, dry, obtains cyclosporin A crude product, total recovery
80.2%, purity 91.3%.
The preparation of 4 cyclosporin A crude product of embodiment
5L cyclosporin A filtering fermentation liquor is obtained into bacteria residue, 65 DEG C drying 4 hours, 2.5L ethyl alcohol is added in the dry bacteria residue of gained
Solution stirs 4 hours, filtering, and 2L ethyl alcohol is added in filter residue and is again stirring for extracting, filters, and merges leaching liquor twice, subtracts at 60 DEG C
Pressure concentration, obtains concentrate.
Into gained concentrate, the NaOH solution of addition 0.5N is about to stir 2 hours under 10,65 DEG C of water-baths to pH, is added
The extraction of 600mL ethyl acetate isolates organic phase, 10g activity carbon decoloring is added in organic phase, stirs 1 hour, filtering, filtrate
It is concentrated under reduced pressure, 70mL acetone is added in residue, stirs evenly, 0.5g cyclosporin A crystal seed is added, is placed under -10 DEG C of environment
Crystallization 20 hours is filtered, dry, obtains cyclosporin A crude product, total recovery 83.3%, purity 91.1%.
Embodiment 5
200L cyclosporin A filtering fermentation liquor is obtained into bacteria residue, 65 DEG C drying 4 hours, 100L second is added in the dry bacteria residue of gained
Alcoholic solution stirs 4 hours, filtering, and 80L ethyl alcohol is added in filter residue and is again stirring for extracting, filters, and merges leaching liquor twice, and 60 DEG C
Under be concentrated under reduced pressure into condensation and ooze, obtain concentrate.
Into gained concentrate, the NaOH solution of addition 1N is about to stir 2 hours under 10,65 DEG C of water-baths to pH, and 20L is added
Ethyl acetate extraction isolates organic phase, 400g activity carbon decoloring is added in organic phase, stirs 1 hour, filtering, filtrate decompression
It is concentrated, 2.9L acetone is added in residue, stirs evenly, 20g cyclosporin A crystal seed is added, places crystallization 20 under -10 DEG C of environment
Hour, it filters, it is dry, obtain cyclosporin A crude product, total recovery 81.2%, purity 91.5%.
Comparative example
5L cyclosporin A fermentation liquid is taken, in addition to base extraction is not added, other operations are found under crystallization condition with embodiment 1
It is generated after placing crystallization 20 hours without crystal, removal acetone is concentrated under reduced pressure and obtains residual crude.
Gained residual crude and 1 gained crude product of embodiment are purified by silica gel column chromatography in the steps below：Dress chromatography is filled out
Expect 160g, loading is eluted with ethyl acetate/petroleum ether mixed solvent, collects the chromatography stream that cyclosporin A component is greater than 98.5%
Part.Product is concentrated under reduced pressure to obtain.Purification result is shown in Table 1.Wherein, upper column capacity is the corresponding cyclosporin A of every gram of chromatographic stuffing
Applied sample amount.
Table 1
| Parameter | 1 gained crude product of embodiment | Residual crude obtained by comparative example |
| Whether crude product crystallizes | It is | It is no |
| Loading capacity (mg/g) | 74 | 25 |
| Applied sample amount (g) | 11.84 | 4.0 |
| Products obtained therefrom quality (g) | 9.72 | 2.45 |
| Yield (%) | 82.1% | 61.3% |
Comparative experiments shows in the concentrate after the extraction of embodiment 1 plus base extraction can remove a large amount of impurity, and extraction is de-
After color can crystallized acquisition crude product, and the concentrate of base extraction is routinely not added in the present embodiment by same purifying process
Afterwards, it can not crystallize.When further through column chromatographic purifying, the upper column capacity of 1 gained crude product of embodiment is far longer than the present embodiment institute
The upper column capacity of residual crude is obtained, the fraction of the cyclosporin A component containing same range is collected, chromatography yield also substantially mentions
It is high.
The preparation of 6 cyclosporin A fine work of embodiment
Fraction of the cyclosporin A component greater than 98.5% that 1 crude product of embodiment is purified by silica gel column chromatography collection is depressurized
Concentration is added acetone solution to 40mg/mL, is placed in -15 DEG C and crystallizes 20 hours.Filtering, 80 DEG C are dried under reduced pressure 7 hours, obtain ring
Spore rhzomorph A fine work, yield 91.3%, purity 99.5%.
It should be pointed out that above-mentioned several preferred embodiments be technical solution of the present invention is made it is further unrestricted detailed
Describe bright, only technical concept and feature to illustrate the invention in detail.Its object is to person skilled in the art can understand
The contents of the present invention are simultaneously implemented accordingly, and it is not intended to limit the scope of the present invention.All institutes of Spirit Essence according to the present invention
The equivalent change or modification of work, should be covered by the protection scope of the present invention.
Claims (9)
1. a kind of preparation method of cyclosporin A crude product, it is characterised in that：Include the following steps：
(1), by cyclosporin A filtering fermentation liquor, bacteria residue is parsed with ethyl alcohol, and concentration removes ethyl alcohol, obtains concentrate；
(2), concentrate adds base extraction to obtain treatment fluid；
(3), treatment fluid is extracted with organic solvent, and decoloration crystallizes to obtain cyclosporin A crude product after concentration.
2. the preparation method of cyclosporin A crude product as described in claim 1, it is characterised in that：Gained crude product purity is 90%
More than.
3. the preparation method of cyclosporin A crude product as described in claim 1, it is characterised in that：The choosing of lye described in step (2)
From NaOH solution, KOH solution or Na2CO3Solution, preferably NaOH solution.
4. the preparation method of cyclosporin A crude product as described in claim 1, it is characterised in that：Lye described in step (2) is dense
Degree is 0.5-1.5N, preferably 1N.
5. such as the preparation method of cyclosporin A crude product of any of claims 1-4, it is characterised in that：In step (2)
Described plus base extraction is plus lye is to system pH=9.5~11.5, and in 50~60 DEG C stirred in water bath 2 hours, preferably 60
℃。
6. the preparation method of cyclosporin A crude product as described in claim 1, it is characterised in that：It is organic molten described in step (3)
Agent is ethyl acetate, and the volume ratio of ethyl acetate and treatment fluid is 3-4: 1.
7. the preparation method of cyclosporin A crude product as described in claim 1, it is characterised in that：It decolourizes described in step (3)
For the active carbon of extract liquor volume 2-4% (W/V) is added, stirs 2 hours, is filtered, washed to obtain destainer.
8. the preparation method of cyclosporin A crude product as described in claim 1, it is characterised in that：Crystallization described in step (3)
Method is that acetone is added in the residue after concentration, and the w/v of residue and acetone is 0.8-1.5: 1, -5 DEG C to -18
It is placed at DEG C crystallization 20 hours or more.
9. the method that cyclosporin A crude product as described in claim 1 prepares high purity cyclosporin A, it is characterised in that：Ring spore
Rhzomorph A crude product collects spore rhzomorph component A and is greater than 98.5% chromatography fraction, concentration through column chromatographic purifying, crystallization obtain purity >=
99.5% cyclosporin A fine work.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HK1007882A1 (en) * | 1995-03-03 | 1999-04-30 | Santen Pharmaceutical Co Ltd | Process for preparation of pure cyclosporin chromatographically using an eluent consisting essentially of high pressure carbon dioxide |
| US6423233B1 (en) * | 2000-08-15 | 2002-07-23 | Biogal Gyogyszergyar Rt. | Purification process |
| CN102086226A (en) * | 2009-12-04 | 2011-06-08 | 山东新时代药业有限公司 | Method for preparing cyclosporine A |
| CN108250275A (en) * | 2016-12-29 | 2018-07-06 | 天津领世生物科技开发有限公司 | A kind of method that cyclosporin A is isolated and purified from zymotic fluid |
-
2018
- 2018-06-28 CN CN201810684846.XA patent/CN108864257A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HK1007882A1 (en) * | 1995-03-03 | 1999-04-30 | Santen Pharmaceutical Co Ltd | Process for preparation of pure cyclosporin chromatographically using an eluent consisting essentially of high pressure carbon dioxide |
| US6423233B1 (en) * | 2000-08-15 | 2002-07-23 | Biogal Gyogyszergyar Rt. | Purification process |
| CN102086226A (en) * | 2009-12-04 | 2011-06-08 | 山东新时代药业有限公司 | Method for preparing cyclosporine A |
| CN108250275A (en) * | 2016-12-29 | 2018-07-06 | 天津领世生物科技开发有限公司 | A kind of method that cyclosporin A is isolated and purified from zymotic fluid |
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| 吴晖: "环孢菌素A菌株选育及生产工艺研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
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Application publication date: 20181123 |