CN108250275A - A kind of method that cyclosporin A is isolated and purified from zymotic fluid - Google Patents
A kind of method that cyclosporin A is isolated and purified from zymotic fluid Download PDFInfo
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- CN108250275A CN108250275A CN201611241709.6A CN201611241709A CN108250275A CN 108250275 A CN108250275 A CN 108250275A CN 201611241709 A CN201611241709 A CN 201611241709A CN 108250275 A CN108250275 A CN 108250275A
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- cyclosporin
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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
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Abstract
The present invention provides a kind of method that cyclosporin A is isolated and purified from zymotic fluid, comprises the steps of：(1) extraction of cyclosporin A；(2) macroporous adsorption resin chromatography；(3) PS30RPC types polymer nano-microspheres column chromatography；(4) crystallization purifying eluent is concentrated in vacuo and crystallizes, and with cold acetone petroleum ether, is finally dried in vacuo to obtain colorless powder cyclosporin A sterling.The fermented liquid extraction of the method for the present invention, nonpolar macroporous adsorption resin chromatography, PS30RPC type polymer nano-microspheres column chromatography, crystallization purifying obtain purity more than 98.5% cyclosporin A, and yield is more than 70%.The present invention is using Isocratic clution, and without complicated intermediate steps solvent replacement process, technological operation is simple and direct, suitable for industrialized production.
Description
Technical field
The present invention relates to biomedicine technical fields, and in particular to a kind of separation purifying technique of cyclosporin A.
Background technology
Cyclosporin A (Ciclosporin A, CsA) is from porous trichoderma (Tolypocladium inflatum
CSP3), one kind isolated in the culture of just entitled Beauveria nivea (Beauveria bassiana) contains 11 ammonia after
The ring type polypeptide of base acid is a kind of very strong immunosuppressor of effect, also has more than 20 kinds of B, C, D etc. other than cyclosporin A together
It is object.For the immunosuppressive therapy of the transfer operations such as kidney, liver and heart, it can also be used to treat ciclosporin A
Autoimmune disease, such as lupus erythematosus, rheumatoid arthritis, Idiopathic Thrombocytopenic Purpura, ulcerative colitis, again
The various autoimmunes disease such as disease myasthenia, chronic nephritis, chronic active hepatitis and nephrotic syndrome.
Major impurity in cyclosporin A fermented product is the similar cyclosporin A homologue of structure, including B, C, D etc.,
It is in the prior art first to be extracted with organic solvent for the extraction purification dominating process route of cyclosporin A, then passes through difference
The column chromatography of type is isolated and purified.
CN200510108097.9 describes one kind and obtains cyclosporin coarse crystal first, recycles column chromatographic isolation and purification
Cyclosporin A, and the purifying process of secondary crystallization is carried out, this method coarse crystallization complex process is more using organic solvent type, has
A little solvents are expensive, and crystallize needs 3 days, and time-consuming for entire purifying process.US5656459 discloses a kind of extraction purification
Technique is first extracted with methanol, and extract is dissolved in ethyl acetate by removal methanol again, and ring spore bacterium is obtained by silica gel column chromatography twice
Plain A sterlings, technique extraction cannot be connected with silica gel column chromatography solvent for use, need to convert, and silica gel column chromatography is often, total to receive
Rate is low.
US5382655 is disclosed a kind of isolates and purifies cyclosporin A by the use of chloroform, dichloromethane and ethyl alcohol as mobile phase
Silica gel column chromatography method, but these solvent toxicities are big, it is unfriendly to environment.Patent US597638 discloses a kind of utilization high pressure two
The method that carbonoxide obtains cyclosporin A as flowing carrier by silica gel column chromatography, but this method is to equipment requirement height, sample
Treating capacity is few, can not realize extensive large-scale production.Patent US2002/0162789A1 discloses a kind of multistep silica gel color
Spectrum separation, mobile phase toluene and ethyl acetate, major defect are that toluene toxicity is big, and chromatographic step is more, and solvent consumption is big.WO01/
64935A1 describe it is a kind of extracted using organic solvent, the side of gel filtration and silica gel column chromatography separating purification cyclosporin A
Method, this method major defect are that gel filtration filler is expensive, and all need to remove the solvent of back between each step,
Heavy workload, solvent consumption are more.US2003/0186856A1 isolates and purifies ring spore bacterium using sephadex by twice chromatographic
The method of plain A, main problem is filler price, of high cost.CA2108655 provides a kind of using superfluid abstraction technique point
Method from cyclosporin A, this method investment is big, of high cost, is not suitable for industrialized production.
Comprehensive disclosure patent is mainly characterized by first being extracted with organic solvent, then utilizes one or multi-step silicagel column
Chromatography is isolated and purified, some separation purifying techniques are of high cost, are not suitable for industrialized production, some process solvent toxicity
Greatly, consumption is big, and total recovery about 40%~60%.
《Chinese antibiotic magazine》Article (number：1001-8689 (2008) 05-0276-04) report is using macroporous absorption
Resin HP combinations high speed adverse current chromatogram detaches cyclosporin small component, can realize that enrichment detaches, but the party again to cyclosporin
Method treatment scale is small, equipment requirement is high, is unfavorable for industrialized production.
Invention content
The purpose of the present invention is to provide a kind of extensive, efficiently industrialization purification high purity cyclosporin A new methods.
It is isolated and purified for cyclosporin A in the prior art in the prevalence of complex process, complex steps, organic solvent kind
The shortcomings such as class is more and dosage is big, and pigment poor removal effect and total recovery are low, inventor to cyclosporin A separation purifying technique into
The a large amount of research of row, for the biochemical property of ciclosporin A, gropes experiment condition and technological parameter, using macroporous absorption and
The separation method that PS30RPC type polymer nano-microspheres column chromatographies are combined, achieves good technique effect, and the present invention uses
Macroporous adsorption resin chromatography technology, organic solvent in extracting solution without displacement can direct loading macroporous absorbent resin, elute
Agent dosage is few, experimentally determined only to need 1.5~2 times of column volumes that elute cyclosporin A completely, and cyclosporin A is played
The enrichment method effect of highly significant, and most pigments can be removed, it solves the disadvantage that original extraction process, improves
Product yield；Since macroporous adsorption resin chromatography has been removed most of impurity, the present invention is in PS30RPC type polymer nanocomposites
Using Non-gradient elution in microballoon column chromatography, and the organic solvent-acetone and water that use toxicity low be as mobile phase, compared to ladder
Degree elution, simplifies chromatography technique, reduces environmental pollution.
The invention is realized by the following technical scheme：
(1) extraction of cyclosporin A
Into zymotic fluid plus NaOH tune PH is to neutrality, adds in organic solvent, stirring extraction 5~8 hours, by extracting solution press filtration
Go out thalline, collect filtrate.
Organic solvent is added in wherein into zymotic fluid to be one or more in methanol, ethyl alcohol and acetone, preferably acetone,
The volume ratio of zymotic fluid and organic solvent is 1: 1~1: 2, preferably 1: 1, after being stirred 5~8 hours under the conditions of 30 DEG C~40 DEG C,
It carries out separation of solid and liquid and collects filtrate.
(2) macroporous adsorption resin chromatography
Macroporous absorbent resin fills column, resin is pre-processed, extracting solution loading, and the tree after loading is handled with detergent
Then fat elution and collects eluent, the eluent of collection is concentrated under reduced pressure, remove organic solvent, adds in extraction
Eluent is taken, extract liquor is concentrated for use.
Cyclosporin A Static Adsorption-desorption experiment is carried out to different model resin HZ816, HP20, H103 and X-5, really
The absorption of HZ816, HP20 and X-5 and desorption excellent effect are determined, wherein more preferable HZ816 resins.
When content of acetone is less than 60% in extracting solution, HZ816 resins are more than the maximal absorptive capacity of cyclosporin A
35mg/g resins, further experiment determine the large amount of adsorption of extracting solution in 10 times of bed volumes；Pre-process detergent used in resin
It is pure water, 5% sodium hydroxide solution and 5% hydrochloric acid solution successively, the resin detergent after loading is followed successively by 5% hydrochloric acid and pure
Water, eluant, eluent are one or more in ethyl acetate, methanol and acetone, and preferably acetone, elution volume are about 1.5~2 times of columns
Bed volume.
(3) PS30RPC types polymer nano-microspheres column chromatography
PS30RPC types polymer nano-microspheres fill column, will extract concentrate loading, are eluted with acetone water mixed solvent, are received
Collect the cyclosporin A eluent of purity qualification.
PS30RPC type polymer nano-microspheres column chromatography selects high mesh number polymer nano-microspheres, 200-300 mesh or 150-
One or both of 200 mesh, preferably 200-300 mesh, PS30RPC type polymer nano-microspheres column chromatography columns ratio of height to diameter is ranging from
8: 1~12: 1, preferably 10: 1, PS30RPC type polymer nano-microspheres column chromatography eluent composition for acetone, ethyl acetate with
Water, proportioning are preferably acetone: ethyl acetate：Water (volume ratio)=60:8:32；When bed volume is 14L, sample solution 10%
Column volume, cyclosporin A sample concentration are 80~110g/L, and preferably 100g/L, eluent flow rate is 70~90mL/min, preferably
80mL/min detects efflux with HPLC, collects qualified products.
(4) crystallization purifying
Eluent is concentrated in vacuo and crystallizes, and is washed with acetone petroleum ether or hexane acetone, is finally dried in vacuo colourless
Powdered cyclosporin A sterling.
Eluent is concentrated under reduced pressure into acetone under the conditions of 40 DEG C~60 DEG C and is all removed with ethyl acetate, adds in 1:1 second
Acetoacetic ester is extracted, and the ethyl acetate containing ciclosporin A is carried out again to be concentrated into 2L, Powdered Activated Carbon 25g is added in, adds anhydrous
Sodium sulphate 50g is stirred decoloration and dry 1h, decoloration is filtered with the dried ethyl acetate containing ciclosporin A, mistake
Ethyl acetate item after filter carries out being concentrated under reduced pressure into yellow lotion again, adds in a small amount of acetone solution, slowly drips under stiring
Add petroleum ether solution, crystallized under the conditions of being placed in -5 DEG C~-20 DEG C, preferably -10 DEG C, crystal is collected by filtration, with cold acetone petroleum ether
Washing is dried under the conditions of 40 DEG C~60 DEG C to constant weight.
The technical effects of the invention are that：By using macroporous adsorption resin chromatography adsorbing separation cyclosporin A, relative to
Traditional extraction technique can make consumption of organic solvent reduce 1-2 times, and pigment removal reaches 95%, and the concentration of cyclosporin A is through dense
Contracting enrichment can improve nearly 10 times, and bulky extractor can be saved from slave device requirement, reduce energy consumption and labour is strong
Degree；PS30RPC type polymer nano-microspheres column chromatographies column chromatography is using the acetone that toxicity is low, price is relatively cheap, ethyl acetate
With water as mobile phase, using Isocratic clution relative to gradient elution, reduce processing step, improve yield (75% with
On) and purity (more than 98% HPLC), in terms of entire process route, without complicated intermediate steps solvent replacement process, technique
It is simple to operation, PS30RPC type polymer nano-microspheres fillers through regeneration can Reusability, suitable for industrialized production.
Description of the drawings
Cyclosporin A crude product HPLC collection of illustrative plates after Fig. 1, macroporous adsorption resin chromatography
The sterling HPLC figures of Fig. 2, PS30RPC type polymer nano-microspheres column chromatography and crystallized rear cyclosporin A
Spectrum
Specific embodiment
Beneficial effects of the present invention are now further described by following embodiment, embodiment is only used for the purpose of illustration,
It does not limit the scope of the invention, while the obvious change and modification that those of ordinary skill in the art are made according to the present invention
It is also contained within the scope of the invention.
Embodiment 1
(1) cyclosporin A zymotic fluid 50L is taken, 5mol/l NaOH is added in and adjusts pH to neutrality, add in 1 times of vol acetone and exist
30 DEG C of stirrings are extracted 8 hours, are filtered off in 1Mp direct draughts except thalline, are collected filtrate and are treated upper prop.
(2) column (Φ 10cm × 120cm) is filled with 5kg HZ816 macroporous absorbent resins, respectively with pure water, 5% sodium hydroxide
Solution and 5% hydrochloric acid solution carrying out washing treatment resin, by the extracting solution upper prop of about 10 times of column volumes after being disposed；With about 2 columns
The salt pickling column of volume 5% is simultaneously washed till pH 6.5~7.5 with pure water；It is eluted with 60% acetone, collects about 1.5 times of column volume elutions
Liquid；Eluent is concentrated under reduced pressure into acetone under the conditions of 50 DEG C and all removes, and adds in 1:1 ethyl acetate is extracted, and decompression is dense
It is reduced to yellow lotion and occurs, the acetone solution with 10% column volume is spare.
(3) by wet method dress post after PS30RPC type polymer nano-microspheres 200-300 mesh proper treatments (Φ 10cm ×
140cm), with 2 column volume acetone: water (volume ratio)=60: 30 balance PS30RPC type polymer nano-microspheres chromatographic columns are put down
The acetone of about 10% column volume is concentrated into reserve liquid upper prop after weighing apparatus, is eluted with acetone/ethyl acetate/water mix reagent, is received
Collect the eluent that purity is higher than 97%.
(4) eluent is concentrated under reduced pressure into acetone under the conditions of 50 DEG C and is all removed with ethyl acetate, adds in 1:1 acetic acid second
Ester is extracted, and the ethyl acetate containing ciclosporin A is carried out again to be concentrated into 2L, Powdered Activated Carbon 25g is added in, adds anhydrous slufuric acid
Sodium 50g is stirred decoloration and dry 1h, decoloration is filtered with the dried ethyl acetate containing ciclosporin A, after filtering
Ethyl acetate item carry out being concentrated under reduced pressure into yellow lotion again, add in a small amount of acetone solution, stone be slowly added dropwise under stiring
Oily ethereal solution crystallizes under the conditions of being placed in -5 DEG C~-20 DEG C, and preferably -10 DEG C, crystal is collected by filtration, with cold acetone petroleum ether
Constant weight is dried under vacuum under the conditions of 50 DEG C, obtains white amorphous powder finished product, HPLC collection of illustrative plates content is calculated up to 98.4%
Total recovery is 71%.
Embodiment 2
(1) cyclosporin A zymotic fluid 50L is taken, 5mol/l NaOH is added in and adjusts pH to neutrality, add in 2 times of volumes methanols and exist
40 DEG C of stirrings are extracted 8 hours, are filtered off in 1Mp direct draughts except thalline, are collected filtrate and are treated upper prop.
(2) it is molten with pure water, 5% sodium hydroxide respectively with 5kg X-5 macroporous absorbent resins dress column (Φ 10cm × 120cm)
Liquid and 5% hydrochloric acid solution carrying out washing treatment resin, after being disposed will about 8 times of column volume extracting solution upper props, with 1.5 times of column volumes
5% salt pickling column is simultaneously washed till pH 6.5~7.5 with pure water, is eluted with ethyl acetate, collects about 2 times of column volume eluents,
The petroleum ether extraction layering of 0.5 times of volume, removal lower floor water phase are added in eluent, concentration upper liquid treats each other upper PS30RPC types
Polymer nano-microspheres column.
(3) this step is the same as 1 step of embodiment (3).
(4) petroleum ether is concentrated under reduced pressure in eluent under the conditions of 60 DEG C and after ethyl acetate, the acetone progress of addition about 2L is molten
Solution adds in Powdered Activated Carbon 25g, and anhydrous sodium sulfate 50g is added to be stirred decoloration and dry 1h, and decoloration is contained ring with dried
The acetone of spore element A is filtered, and the acetone item after filtering carries out being concentrated under reduced pressure into the appearance of yellow lotion again, adds in a small amount of third
Ketone dissolves, and petroleum ether solution is slowly added dropwise under stiring, is crystallized under the conditions of being placed in -5 DEG C~-20 DEG C, preferably -10 DEG C, and filtering is received
Collect crystal, constant weight be dried under vacuum under the conditions of 50 DEG C with cold acetone petroleum ether, obtains white amorphous powder finished product,
It is 69% that HPLC collection of illustrative plates content calculates total recovery up to 98.1%.
Embodiment 3
(1) cyclosporin A zymotic fluid 50L is taken, 5mol/l NaOH is added in and adjusts pH to neutrality, add in 1.5 times of volumes of acetic acid
Ethyl ester extracts 6 hours in 35 DEG C of stirrings, and -0.06Mpa vacuum filtration removal thalline collect filtrate and treat upper prop.
(2) it is molten with pure water, 5% sodium hydroxide respectively with 5kg HP20 macroporous absorbent resins dress column (Φ 10cm × 120cm)
Liquid and 5% hydrochloric acid solution carrying out washing treatment resin, will about 7 times of column volume extracting solution upper props after being disposed；With 2 times of column volumes 5%
Salt pickling column and be washed till pH 6.5~7.5 with pure water；It is eluted with methanol, collects about 1.5 times of column volume eluents；In eluent
The middle petroleum ether extraction layering for adding in 0.5 times of volume, removal lower floor water phase, concentration upper liquid treat each other upper prop.
(3) this step is the same as 1 step of embodiment (3).
(4) petroleum ether is concentrated under reduced pressure in eluent under the conditions of 60 DEG C and after ethyl acetate, the acetone progress of addition about 2L is molten
Solution adds in Powdered Activated Carbon 25g, and anhydrous sodium sulfate 50g is added to be stirred decoloration and dry 1h, and decoloration is contained ring with dried
The acetone of spore element A is filtered, and the acetone item after filtering carries out being concentrated under reduced pressure into the appearance of yellow lotion again, adds in a small amount of third
Ketone dissolves, and hexane solution is slowly added dropwise under stiring, is crystallized under the conditions of being placed in -5 DEG C~-20 DEG C, preferably -10 DEG C, and filtering is received
Collect crystal, washed with cold acetone n-hexane and constant weight is dried under vacuum under the conditions of 50 DEG C, obtain white amorphous powder finished product,
It is 67% that HPLC collection of illustrative plates content calculates total recovery up to 98.3%.
Claims (8)
- A kind of 1. method that cyclosporin A is isolated and purified from zymotic fluid, it is characterised in that：Its process comprises the steps of：(1) extraction of cyclosporin AInto zymotic fluid plus NaOH tune pH value is neutrality, adds in organic solvent, and extracting solution is filtered out bacterium by stirring extraction 5~8 hours Body collects filtrate, and the organic solvent is one or more in methanol, ethyl alcohol and acetone；(2) macroporous adsorption resin chromatographyColumn is filled with the nonpolar macroporous adsorption resin of model HZ816, HP20, X-5 or H103, resin is pre-processed, is carried Liquid loading is taken, the resin after loading is handled with detergent, then elution and collects eluent, it is organic that removal is concentrated under reduced pressure It is organic that removal is concentrated under reduced pressure in the aqueous solution of solvent or reduced pressure removal organic solvent addition ethyl acetate extraction containing ciclosporin A again It is for use to obtain concentrate for solvent；(3) PS30RPC types polymer nano-microspheres column chromatographyPS30RPC types polymer nano-microspheres are filled into column, concentrate loading, with acetone, the mixing of ethyl acetate and water will be extracted Solvent elutes, and collects the cyclosporin A eluent of purity qualification；(4) crystallization purifyingEluent is concentrated in vacuo and crystallizes, and with cold acetone petroleum ether, is finally dried in vacuo to obtain colorless powder cyclosporin A Sterling.
- A kind of 2. method that cyclosporin A is isolated and purified from zymotic fluid according to claim 1, it is characterised in that step (1) volume ratio of zymotic fluid and organic solvent is 1:1~1:2；Step (1) organic solvent is acetone；Step (1) broth extraction Temperature is 30 DEG C~40 DEG C.
- A kind of 3. method that cyclosporin A is isolated and purified from zymotic fluid according to claim 1, it is characterised in that step (2) big pore adsorption resin model is HZ816；Detergent used in step (2) pretreatment resin is pure water, 5% hydrogen successively Sodium hydroxide solution and 5% hydrochloric acid solution；Resin detergent after step (2) loading is 50-60% acetone solns；Step (2) is washed De- agent is one or more in ethyl acetate, methanol and acetone.
- A kind of 4. method that cyclosporin A is isolated and purified from zymotic fluid according to claim 1, it is characterised in that step (3) the selected granularity of macroreticular resin chromatography is one or both of 200-300 mesh and 100-200 mesh；Step (3) macropore tree Fat column chromatography column ratio of height to diameter is 8:1~12:1；Step (3) PS30RPC type polymer nano-microspheres column chromatography column ratio of height to diameters are preferred 10:1；Step (3) sample solution is 10%~30% column volume；Step (3) sample solution is 10% column volume；Step (3) macropore tree Eluent proportioning is volume ratio acetone in fat column chromatography:Water=50~62：50~38；Step (3) PS30RPC type polymer nanocomposites Eluent proportioning is preferably volume ratio acetone in microballoon column chromatography:Ethyl acetate：Water=60：8：32.
- A kind of 5. method that cyclosporin A is isolated and purified from zymotic fluid according to claim 1, it is characterised in that step (4) macroporous resin column chromatography eluent temperature concentrated in vacuo is 40 DEG C~60 DEG C.
- A kind of 6. method that cyclosporin A is isolated and purified from zymotic fluid according to claim 1, it is characterised in that step (4) crystallization temperature of cyclosporin A is -5 DEG C~-20 DEG C.
- A kind of 7. method that cyclosporin A is isolated and purified from zymotic fluid according to claim 1, it is characterised in that step (4) crystallization temperature of cyclosporin A is -10 DEG C.
- A kind of 8. method that cyclosporin A is isolated and purified from zymotic fluid according to claim 1, it is characterised in that step (4) vacuum drying temperature of cyclosporin A is 40 DEG C~60 DEG C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864257A (en) * | 2018-06-28 | 2018-11-23 | 杭州中美华东制药有限公司 | High purity cyclosporin A and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5382655A (en) * | 1992-05-25 | 1995-01-17 | Biogal Gyogyszerarugyar Rt | Process for the purification of cyclosporin A |
| EP0725076A1 (en) * | 1995-02-01 | 1996-08-07 | National Research Development Corporation of India | A process for the preparation of cyclosporin A from tolypocladium species |
| CN102086226A (en) * | 2009-12-04 | 2011-06-08 | 山东新时代药业有限公司 | Method for preparing cyclosporine A |
| CN103073624A (en) * | 2011-10-25 | 2013-05-01 | 华北制药集团新药研究开发有限责任公司 | Preparation method of high-purity cyclosporin A derivative |
-
2016
- 2016-12-29 CN CN201611241709.6A patent/CN108250275A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5382655A (en) * | 1992-05-25 | 1995-01-17 | Biogal Gyogyszerarugyar Rt | Process for the purification of cyclosporin A |
| EP0725076A1 (en) * | 1995-02-01 | 1996-08-07 | National Research Development Corporation of India | A process for the preparation of cyclosporin A from tolypocladium species |
| CN102086226A (en) * | 2009-12-04 | 2011-06-08 | 山东新时代药业有限公司 | Method for preparing cyclosporine A |
| CN103073624A (en) * | 2011-10-25 | 2013-05-01 | 华北制药集团新药研究开发有限责任公司 | Preparation method of high-purity cyclosporin A derivative |
Non-Patent Citations (3)
| Title |
|---|
| M. V. RAMANA MURTHY,ET AL.: "Cyclosporin-A production by Tolypocladium inflatum using solid state fermentation", 《PROCESS BIOCHEMISTRY》 * |
| 任风芝等: "环孢素衍生物(γ-hydroxylmethylleucine4)-cyclosporin A的分离纯化与鉴定", 《中国抗生素杂志》 * |
| 董惠钧等: "大孔吸附树脂结合硅胶柱层析纯化环孢菌素A", 《生物加工过程》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864257A (en) * | 2018-06-28 | 2018-11-23 | 杭州中美华东制药有限公司 | High purity cyclosporin A and preparation method thereof |
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