CN109553646A - A kind of preparation method purifying Calicheamicin - Google Patents
A kind of preparation method purifying Calicheamicin Download PDFInfo
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- CN109553646A CN109553646A CN201910055747.XA CN201910055747A CN109553646A CN 109553646 A CN109553646 A CN 109553646A CN 201910055747 A CN201910055747 A CN 201910055747A CN 109553646 A CN109553646 A CN 109553646A
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- calicheamicin
- concentrate
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- organic solvent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
Abstract
The invention discloses a kind of purifying Calicheamicin γ1 IMethod.This method comprises the following steps: (1) by Calicheamicin γ1 ICrude product carries out positive and prepares column chromatography, is eluted with organic solvent, collects and merge containing Calicheamicin γ1 IEluent；(2) the isolated Calicheamicin γ from eluent obtained by step (1)1 IHigh purity extractive product.Method of the invention can obtain the Calicheamicin γ of the high-purity of purity >=99%1 I, and high income, it is environmental-friendly, it is very suitable to commercially produce.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to antibiotic Calicheamicin γ1 IPurification process.
Background technique
Calicheamicin (Calicheamicin) is a kind of small molecule fat-soluble compound, belongs to ten-ring Enediyne
Antitumor antibiotics is from one plant of rare actinomycete micromonospora Micromonospora echinospora spp
Isolated in the fermentation liquid of calichensis, main active is Calicheamicin γ1 I.Calicheamicin has
Very strong cytotoxicity and anti-tumor activity can cause DNA double chain fracture to kill in the ditch of intercalation of DNA double helix
Cell.Calicheamicin has strong lethal effect to kinds of tumor cells, is the activity strongest one found so far
Class antitumor antibiotics.Since Calicheamicin antitumor action is extremely strong, molecule is smaller, by as a kind of monoclonal antibody
" bullet " is applied to clinic.
Calicheamicin γ1 IStructural formula it is as follows:
In relation to reporting that the document of Calicheamicin has American Chemical Society (J.Am.Chem.Soc., 1987,109:3464-
3466；J.Am.Chem.Soc.,1987,109:3466-3468)；Japanese antibiotic magazine (J.Antibiotics., 1989,
42:558-563；J.Antibiotics.,1989,42:1070-1087.)；And Chinese patent CN101591632A etc..These
The document report physicochemical property of Calicheamicin, bioactivity, the contents such as ferment and isolate and purify.The prior art is not directed to
Large scale preparation obtains the Calicheamicin γ of 99% or more purity1 I, therefore be badly in need of a kind of can be prepared on a large scale, high-purity
Calicheamicin γ1 IPreparation method, comply with pharmaceutical production requirement.
Summary of the invention
It is an object of the invention to provide a kind of environmental-friendly, simple process, the purifying card Ritchies that can implement on a large scale
Mycin γ1 IMethod, the purpose of the present invention can be achieved through the following technical solutions:
A kind of purifying Calicheamicin γ1 IMethod, described method includes following steps:
(1) by Calicheamicin γ1 ICrude product carries out positive and prepares column chromatography, is eluted with organic solvent, collects and close
And contain Calicheamicin γ1 IEluent；
(2) the isolated Calicheamicin γ from eluent obtained by step (1)1 IHighly finished product.
Wherein, the column packing for preparing that positive described in step (1) prepares column chromatography composes the positive phase filling of X5 for China, and Hua Pu X5 is just
Phase filling is a kind of polynary alcohol radical bonded-phase silica of spherical shape that can be reused.Organic solvent described in step (1) is acetic acid second
Ester-methanol solvate system or methylene chloride-methanol solvent system, preferably acetate-methanol=100:1-10 (V:V), or
The solution of methylene chloride-methanol=100:1-10 (V:V), the more preferably solution of methylene chloride-methanol=100:3-6 (V:V).
Wherein, it in step (1), collects and merges Calicheamicin γ1 IThe eluent of HPLC purity >=99%.
Wherein, step (1) the Calicheamicin γ1 ICrude product is prepared by following methods:
(a) Calicheamicin γ will be contained1 IFermentation liquid pre-processed, obtain containing Calicheamicin γ1 IIt is molten
Liquid；
(b) by Calicheamicin γ obtained by step (a)1 ISolution be concentrated, obtain containing Calicheamicin γ1 I's
Concentrate 1；
(c) concentrate 1 obtained by step (b) is obtained after cleaning solution washs containing Calicheamicin γ1 IOrganic phase, will
The organic phase continues to be concentrated to get concentrate 2；
(d) the obtained concentrate 2 of step (c) is precipitated by precipitation solvent, and Calicheamicin γ is obtained by filtration1 ICrude product.
Wherein, pretreatment described in step (a) will be the following steps are included: will contain Calicheamicin γ1 IFermentation liquid use
The organic solvent of 0.5-2 times of fermentating liquid volume is mixed, and the organic solvent is ethyl acetate, butyl acetate or acetic acid isopropyl
Ester, ethyl acetate；Then it is layered, is obtained containing Calicheamicin γ after resulting mixture being centrifuged or filtered1 I
Organic solution.
Wherein, it is single tank concentration or scraper plate concentration that the method used is concentrated described in step (b), is concentrated into original volume
One third is to a quarter.
Wherein, in step (c), the cleaning solution is 0.5%kg/L sodium hydroxide solution or 2-5%kg/L sodium carbonate
Solution, preferably 2-5%kg/L sodium carbonate liquor；The volume of the cleaning solution and the volume ratio of concentrate 1 are 0.5-2:1；Institute
The volume ratio of the concentrate 1 and concentrate 2 stated is 10-20:1.
Wherein, in step (d), the precipitation solvent is normal heptane or n-hexane, preferably n-hexane；The precipitating is molten
The volume of agent is 3-5 times of 2 volume of concentrate.
Present invention has an advantage that
The present invention is in Calicheamicin γ1 IIn the preparation process of crude product, Calicheamicin γ will be contained1 IFermentation liquid adopt
After extraction, washing, precipitating, content in fermentation liquid can be only had to the Calicheamicin γ of 20-60mg/L1 IIt is increased to content
70% or more；The present invention provides the method for preparing column chromatographic purifying Calicheamicin using positive, this method selects China spectrum X5
Positive phase filling (i.e. spherical polynary alcohol radical bonded-phase silica) prepares column, so that impurity and Calicheamicin γ1 IEffectively divided
From obtained Calicheamicin γ1 IHPLC purity >=99%, prepares yield >=90%.In addition, organic solvent used in the present invention can
Recycling is reused, and three waste discharge is few, environmental-friendly, meets current medical Production trend and requirement；In addition, this method technique letter
It is single, it is the purifying Calicheamicin γ that can implement on a large scale1 IMethod, meet pharmaceutical production requirement.
Detailed description of the invention
Calicheamicin γ in Fig. 1 embodiment 11 IFermentation liquid HPLC map
The Calicheamicin γ that Fig. 2 embodiment 1 is prepared1 ICrude product HPLC map
The Calicheamicin γ that Fig. 3 embodiment 1 is prepared1 IHighly finished product HPLC map
Specific embodiment
Column is prepared used in the present invention purchased from Beijing Chuangxin Tongheng Science and Technology Co., Ltd.；China's spectrum positive phase filling of X5 is purchased from Zhejiang China
Pu Xinchuan Science and Technology Ltd.；High performance liquid chromatograph is Shimadzu LC2010HT；Disk centrifuge is that the huge energy in Jiangsu is mechanical limited
Company DHC211.Prepare Calicheamicin γ1 IEthyl acetate used in crude product, sodium carbonate are commercially available technical grade；Preparation methanol,
Methylene chloride is analysis level.
Production Calicheamicin γ in preparation example of the present invention1 IBacterial strain is that micromonospora echinospora M.echinospora exists
It is disclosed in CN102732580A, the public can obtain from Haizheng Medicine Stock Co., Ltd., Zhejiang Prov.
The present invention measures Calicheamicin γ1 IContent and the method for chromatographic purity use high performance liquid chromatography, specific side
Method is as follows: Shimadzu LC2010HT liquid phase systems and Agilent ZORBAX SB-C18 (5um, 250 × 4.6mm) chromatographic column, flowing
It is mutually 0.01mol/L ammonium formate: 95% acetonitrile=45:55 (V/V), Detection wavelength 230nm, elution flow rate 1ml/min.Into
Sample amount is 10 μ L.
Preparation example:
Fermentation liquid incubation of the present invention is as follows:
(1) preparation and culture of flat-plate bacterial colony
Plate uses ISP2 culture medium (g/L): glucose 4, yeast extract 4, malt extract 10, agar 20, distillation
Water 1000mL, pH7.0, sterilization pressure 1.05kg/cm2, 20min is cooled to 50-60 DEG C of inverted plate.Single colonie is separated, through 28 ±
1 DEG C culture 5-7 days after dibbling, 27-29 DEG C of plate culture 8-13 days of dibbling.
(2) preparation of shake-flask seed liquid and culture seed culture based formulas (g/L): sucrose 4, mannitol 14, yeast extract 6,
Yeast extract 6, calcium carbonate 4, pH 7.0, shaking flask liquid amount are 25mL/250mL, are sterilized 30 minutes through 121 DEG C.Picking single bacterium
It falls within concussion in 2.0ml bead water pipe to break up (about 10-15min), every bottle of seed accesses 1/2 single colonie, and (i.e. 1ml bacterium is outstanding
Liquid), seed is placed on (240-260) rev/min shaking table, is cultivated (40-48) hour in (26-30) DEG C, pH about 6.8-7.3, and bacterium is dense
14-22%, viscosity (16s) about 120cps.
(3) preparation of seeding tank seed liquor
Seed culture medium (the culture medium prescription (g/L): sucrose 4, mannitol 14, ferment of 100L is put into 120L seeding tank
Female cream 6, yeast extract 6, calcium carbonate 4, pH 7.0 sterilize and use steam sterilizing, 30 minutes under the conditions of 121 DEG C, after cooling,
1-6L shake-flask seed liquid is accessed, cultivation temperature is 28 ± 2 DEG C, speed of agitator 200rpm, air mass flow 1.0-3.5m3/h, culture
40-96 hours
(4) preparation and culture of fermentation tank culture medium
Fermentative medium formula (g/L): sucrose 5, maltodextrin 12, dregs of beans 10, yeast extract 6, soy peptone 10, soya-bean oil
4, bitter salt 0.2, calcium carbonate 4, resin 30, pH 7.0, the volume that feeds intake is 1200L, pH7.0, is steamed under the conditions of 121 DEG C
Vapour sterilizes 20 minutes, after cooling, access about 100L seed tank culture liquid, and 28 ± 2 DEG C of fermentation temperature, speed of agitator 200-
300rpm, ventilatory capacity 140m3/h or more fermented and cultured 8 days, obtain γ containing Calicheamicin1 IFermentation liquid.
Embodiment 1:
The 1200L Calicheamicin γ that will be fermented by the method for preparation example1 IFermentation unit be 29mg/L fermentation
1200L ethyl acetate, plate-frame filtering after stirring 2 hours, obtained filtrate stratification is added in liquid (its chromatograms is shown in attached drawing 1)
Afterwards, it obtains containing Calicheamicin γ1 IEthyl acetate solution.After ethyl acetate solution is concentrated into 300L, 150L is added
After the washing of 3%kg/L sodium carbonate liquor, ethyl acetate solution continues to be concentrated into 25L, and 125L n-hexane precipitating is added, is obtained by filtration
Calicheamicin γ1 ICrude product, content 75.7%.(chromatograms are shown in attached drawing 2).
Column, dichloromethane are prepared using Beijing Chuangxin Tongheng Science and Technology Co., Ltd. DAC200 equipped with China's spectrum positive phase filling of X5
It upper prop and is eluted after alkane-methanol=100:5 (V:V) solution above-mentioned crude product of flowing phased soln, collects HPLC purity >=99%
Component merges the component of purity >=99%, is concentrated and dried, obtains Calicheamicin γ1 IDry powder 75g, 99.3% (its of chromatographic purity
Chromatograms are shown in attached drawing 3), prepare yield 91.2%.
Embodiment 2:
The 1100L Calicheamicin γ that will be fermented by the method for preparation example1 IFermentation unit is the fermentation liquid of 23mg/L
1100L ethyl acetate is added, is centrifuged (revolving speed 6500r/min) with disk centrifuge after stirring 2 hours, obtains mould containing card Ritchie
Plain γ1 IEthyl acetate solution.After ethyl acetate solution is concentrated into 300L, the washing of 300L 3%kg/L sodium carbonate liquor is added
Afterwards, ethyl acetate solution continues to be concentrated into 20L, and 100L n-hexane precipitating is added, Calicheamicin γ is obtained by filtration1 ICrude product contains
Amount 72.3%.
Column, dichloromethane are prepared using Beijing Chuangxin Tongheng Science and Technology Co., Ltd. DAC200 equipped with China's spectrum positive phase filling of X5
It upper prop and is eluted after alkane-methanol=100:6 (V:V) solution above-mentioned crude product of flowing phased soln, collects HPLC purity >=99%
Component merges the component of purity >=99%, is concentrated and dried, obtains Calicheamicin γ1 IDry powder 51g, chromatographic purity 99.1%, system
Standby yield 92.5%.
Above embodiments, which are used merely to explain, realizes method of the invention, should not be construed as limiting the invention, based on this
The modification of invention all belongs to the scope of protection of the present invention.
Claims (10)
1. a kind of purifying Calicheamicin γ1 IMethod, which is characterized in that described method includes following steps:
(1) by Calicheamicin γ1 ICrude product carries out positive and prepares column chromatography, is eluted with organic solvent, collects and merge and contain
There is Calicheamicin γ1 IEluent；
(2) the isolated Calicheamicin γ from eluent obtained by step (1)1 IHighly finished product.
2. the method according to claim 1, wherein step (1) the Calicheamicin γ1 ICrude product is by following
What method was prepared:
(a) Calicheamicin γ will be contained1 IFermentation liquid pre-processed, obtain containing Calicheamicin γ1 ISolution；
(b) by Calicheamicin γ obtained by step (a)1 ISolution be concentrated, obtain containing Calicheamicin γ1 IConcentrate
1；
(c) concentrate 1 obtained by step (b) is obtained after cleaning solution washs containing Calicheamicin γ1 IOrganic phase, this is had
Machine mutually continues to be concentrated to get concentrate 2；
(d) the obtained concentrate 2 of step (c) is precipitated by precipitation solvent, and Calicheamicin γ is obtained by filtration1 ICrude product.
3. according to the method described in claim 2, it is characterized in that, pretreatment described in step (a) will be the following steps are included: will contain
There is Calicheamicin γ1 IFermentation liquid mixed with the organic solvent of 0.5-2 times of fermentating liquid volume, the organic solvent be second
Acetoacetic ester, butyl acetate or isopropyl acetate, ethyl acetate；Then divide after resulting mixture being centrifuged or filtered
Layer, obtains containing Calicheamicin γ1 IOrganic solution.
4. according to the method in claim 2 or 3, which is characterized in that it is single tank that the method used is concentrated described in step (b)
Concentration or scraper plate concentration, are concentrated into the one third of original volume to a quarter.
5. according to the described in any item methods of claim 2-4, which is characterized in that in step (c), the cleaning solution is
0.5%kg/L sodium hydroxide solution or 2-5%kg/L sodium carbonate liquor, preferably 2-5%kg/L sodium carbonate liquor；Described washes
The volume ratio of the volume and concentrate 1 of washing liquid is 0.5-2:1；The volume ratio of the concentrate 1 and concentrate 2 is 10-20:1.
6. according to the described in any item methods of claim 2-5, which is characterized in that in step (d), the precipitation solvent is positive
Heptane or n-hexane, preferably n-hexane；The volume of the precipitation solvent is 3-5 times of 2 volume of concentrate.
7. method according to claim 1-6, which is characterized in that positive described in step (1) prepares column chromatography
Prepare column packing for China spectrum the positive phase filling of X5.
8. method according to claim 1-7, which is characterized in that organic solvent described in step (1) is acetic acid
Ethyl ester-methanol solvate system or methylene chloride-methanol solvent system.
9. method according to claim 1-8, which is characterized in that organic solvent described in step (1) is acetic acid
The solution of ethyl ester-methanol=100:1-10 (V:V) or methylene chloride-methanol=100:1-10 (V:V), preferably methylene chloride-first
The solution of alcohol=100:3-6 (V:V).
10. -9 described in any item methods according to claim 1, which is characterized in that in step (1), collect and merge card Ritchie
Mycin γ1 IThe eluent of HPLC purity >=99%.
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| CN201910055747.XA CN109553646A (en) | 2019-01-21 | 2019-01-21 | A kind of preparation method purifying Calicheamicin |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0182152A2 (en) * | 1984-11-16 | 1986-05-28 | American Cyanamid Company | Antitumor antibiotics (LL-E33288 Complex) |
| CN101591632A (en) * | 2009-04-30 | 2009-12-02 | 中国医学科学院医药生物技术研究所 | One strain produces the micromonospora C-3509 of antitumor antibiotics Cali mycin |
| CN102747115A (en) * | 2011-04-19 | 2012-10-24 | 上海医药工业研究院 | Fermentation medium for fermentation production of antibiotic calicheamicin gamma1 I, fermentation method suitable for the fermentation medium and use of the fermentation medium |
-
2019
- 2019-01-21 CN CN201910055747.XA patent/CN109553646A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0182152A2 (en) * | 1984-11-16 | 1986-05-28 | American Cyanamid Company | Antitumor antibiotics (LL-E33288 Complex) |
| CN101591632A (en) * | 2009-04-30 | 2009-12-02 | 中国医学科学院医药生物技术研究所 | One strain produces the micromonospora C-3509 of antitumor antibiotics Cali mycin |
| CN102747115A (en) * | 2011-04-19 | 2012-10-24 | 上海医药工业研究院 | Fermentation medium for fermentation production of antibiotic calicheamicin gamma1 I, fermentation method suitable for the fermentation medium and use of the fermentation medium |
Non-Patent Citations (1)
| Title |
|---|
| MAY D. LEE，等: "CALICHENAMICINS, A NOVEL FAMILY OF ANTITUMOR ANTIBIOTICS 3. ISOLATION, PURIFICATION AND CHARACTERIZATION OF CALICHEAMICINS β1Br, γ1Br, α2I, α3I, β1I, γ1I, AND δ1I", 《THE JOURNAL OF ANTIBIOTICS》 * |
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Application publication date: 20190402 |