WO2016004848A1 - 一种纯化非达霉素的方法 - Google Patents

一种纯化非达霉素的方法 Download PDF

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WO2016004848A1
WO2016004848A1 PCT/CN2015/083435 CN2015083435W WO2016004848A1 WO 2016004848 A1 WO2016004848 A1 WO 2016004848A1 CN 2015083435 W CN2015083435 W CN 2015083435W WO 2016004848 A1 WO2016004848 A1 WO 2016004848A1
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fidamycin
damycin
water
organic solvent
concentrate
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PCT/CN2015/083435
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English (en)
French (fr)
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任会军
李道超
应雪肖
陈�峰
郑玲辉
王玲萍
白骅
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浙江海正药业股份有限公司
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Priority to JP2017500824A priority Critical patent/JP6592500B2/ja
Priority to US15/324,555 priority patent/US10316052B2/en
Priority to EP15818964.7A priority patent/EP3168225A4/en
Publication of WO2016004848A1 publication Critical patent/WO2016004848A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Definitions

  • the invention belongs to the technical field of medicine, and particularly relates to a method for purifying antibiotic Fidamycin.
  • Fidaxomicin is a novel narrow-spectrum macrolide antibacterial drug developed by Op-timer Pharmaceuticals in recent years. It was approved by the FDA on May 27, 2011 and is available in the US under the trade name Dificid. The drug is mainly used for the treatment of Clostridium difficile (also known as Clostridium difficile) associated diarrhea (CDAD).
  • CDAD Clostridium difficile
  • the currently disclosed methods for separation and purification of Fidamycin mainly include:
  • CN103275152A discloses a preparation method of high-purity fidelity, which comprises filtering a non-damycin fermentation broth to obtain a mycelium, and then immersing the mycelium with a polar solvent and separating the solid and liquid to obtain a non-damycin immersion solution.
  • the liquid is extracted, and the extract is diluted with water and then introduced into a large-pore decolorizing resin to obtain a decolorizing liquid.
  • the decolorizing liquid is introduced into the macroporous adsorption resin to be saturated, and then eluted with a gradient of a resolving agent, concentrated, extracted, and dried.
  • the crude crude drug is dissolved in a polar solvent and then injected into a polymer microsphere column chromatography.
  • the elution solution is eluted with an eluent.
  • the eluate is collected and the HPLC is used to determine the elution of the nondamycin content greater than 95%.
  • CN101663312A discloses a preparation method of pentamycin which adds an absorbent resin to a fermentation broth medium, and after the fermentation is completed, separates a solid substance (including an absorbent resin) from the fermentation broth, and uses an organic solvent such as acetic acid. The solid was eluted with ethyl acetate and then concentrated under reduced pressure to give crude crude. Purification was carried out using a Biotage KP-C 18-HS silica column, followed by concentration, crystallization, and drying to give a purity of 78% to 94.7% of the drug.
  • CN102993251A discloses a method for purifying fidelity by high performance liquid chromatography, which purifies a crude product of 98% pure Fidamycin by two Uni30BPCs to obtain a fidelity eluate having a purity of 98%.
  • WO2011146621A2 discloses a method for preparing a non-damycin which is prepared in a liquid phase to obtain a nondamycin having a purity of about 93%.
  • the purity of the Fudamycin prepared by the above method is below 99%, and still cannot meet the requirements for drug production. Therefore, a preparation method of high-purity Fidamycin is urgently required to meet the requirements for drug production.
  • the object of the present invention is to provide an environmentally friendly and simple process for purifying fidelity, and the object of the present invention can be achieved by the following technical solutions:
  • a method of purifying Fidamycin comprising the steps of:
  • a non-damycin derivative is isolated from the eluate obtained in the step (1).
  • the preparation column described in the step (1) can be a preparative column conventionally in the art, generally a reverse phase chromatography column, and the model of the invention is DAC200 preparative column produced by Beijing Innovation Tongheng Technology Co., Ltd. (200 ⁇ 250mm).
  • the filler for preparing the column may be a conventional filler in the art, preferably a C8 filler.
  • the C8 filler refers to an octylsilane-bonded silica gel, which is generally commercially available in the art and has a particle size. Generally 10 ⁇ m, a Huashi C8 filler, a nano-C8 filler, an Aijieer C8 filler or a kromasil C8 filler, preferably a kromasil C8 filler, may be used.
  • the organic solvent described in the step (1) may be a conventional organic solvent in the art, preferably methanol or acetonitrile; and the aqueous acid solution is an aqueous solution of formic acid.
  • the high performance liquid chromatograph may be a chromatograph conventional in the art, preferably Tsushima
  • the company's model is LC2010HT high performance liquid chromatography.
  • the column temperature can be a conventional column temperature in the art, preferably 20 to 30 °C.
  • the detection wavelength is generally 250 nm.
  • the flow rate is generally 1.0 mL/min.
  • the injection volume is typically 10 uL.
  • the eluate of the nondamycin HPLC purity ⁇ 99.5% is collected and combined.
  • the crude Fidamycin according to the step (1) can be prepared according to a conventional preparation method in the art, and generally requires that the HPLC purity of the crude Fudamycin is above 70%, for example, 72.6%, 70.9%, 72% or 73.5. %, that's it.
  • the crude Fidamycin is prepared by the following method:
  • step (b) concentrating the solution of the non-damycin obtained in the step (a) by nanofiltration to obtain a concentrate containing the Fidamycin;
  • the fermentation liquid containing Fidamycin can be as conventional in the art.
  • the preparation method is prepared.
  • the invention preferably includes the following methods:
  • the shake flask seed liquid obtained in the step 2 is inoculated into the seed tank seed culture medium to obtain a seed tank culture liquid;
  • the seed tank culture solution prepared in the step 3 is inoculated into a fermentation medium to obtain a fermentation liquid containing Fidamycin.
  • the Fidamycin-producing biological bacteria generally refers to a biological bacteria capable of producing Fidamycin after fermentation, and the Actinoplanes sp. HS-16-20 is preferred in the present invention. It was deposited on March 11, 2013 at the General Microbiology Center of the China Microbial Culture Collection Management Committee. The deposit address is: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the strain collection number is CGMCC NO. 7294, classified as Actinoplanes sp., and registered for survival.
  • the plate medium may be a plate medium conventional in the art, preferably an ISP2 medium.
  • the ISP2 medium preferably comprises glucose 4 g/L, yeast extract 4 g/L, malt extract 10 g/L, agar 15 g/L, and the balance is water, wherein g/L refers to 1 L medium.
  • the pH in the ISP2 medium is preferably 7.3.
  • the plate medium is generally sterilized prior to use.
  • the method of sterilization described can be a method conventional in the art.
  • the sterilization pressure is preferably 1.05 kg/cm 2 .
  • the sterilization time is preferably 20 min.
  • the temperature of the culture may be a temperature conventional in the art, preferably from 27 to 29 ° C, more preferably 28 ° C.
  • the culture time may be a routine time in the art as long as the hyphamycin-producing fungus hyphae can be matured, preferably 8 days.
  • the shake flask seed culture medium can be a conventional shake flask seed culture medium, preferably, including sucrose 2 g/L, sorbitol 3 g/L, cotton seed cake powder 3 g/L, peanut cake powder. 1.5 g/L, CaCO 3 0.6 g/L, MgSO 4 ⁇ 7H 2 O 0.3 g/L, and the balance is water, wherein g/L means the mass of each component in the 1 L seed medium.
  • the pH of the shake flask seed medium is preferably 7.2.
  • the shake flask seed culture medium is generally sterilized prior to use.
  • the method of sterilization described can be a method conventional in the art.
  • the sterilization temperature is preferably 121 °C.
  • the sterilization time is preferably 30 min.
  • the inoculum amount of the Fidamycin-producing biological bacterial colonies may be a conventional inoculum amount in the art, and preferably the inoculum amount of the Fudamycin-producing biological bacteria per ml (mL) of the shake flask seed culture medium is 10 5 to 10 6 cfu.
  • the temperature of the culture may be a temperature conventional in the art, preferably from 27 to 29 ° C, more preferably 28 ° C.
  • the manner of culturing can be in a conventional manner in the art, preferably shaking shake culture.
  • the rotation speed of the culture is preferably 250 rpm.
  • the culture time may be a conventional culture time in the art, preferably 28 hours.
  • the pH of the obtained shake flask seed solution is generally between 6.8 and 7.0.
  • the concentration of the fibrin-producing fungus hyphae in the shake flask seed liquid is generally 25% to 30%, and the percentage refers to the volume of the fibrin-producing fungus hyphae as the shake flask seed. The percentage of the volume of the liquid.
  • the seed tank seed culture medium may be a seed seed seed culture medium conventional in the art, preferably including sucrose 10 g/L, sorbitol 2 g/L, soluble starch 3 g/L, (NH 4 ) 2 . SO 4 0.5g / L, beef extract 2g / L, peanut cake powder 1g / L, KH 2 PO 4 0.04g / L, the rest is water, wherein g / L refers to the components in the 1L seed tank medium the quality of.
  • the seed tank seed culture medium is generally sterilized prior to use.
  • the method of sterilization can be a conventional method in the art, preferably steam sterilization.
  • the temperature of the steam sterilization may be a temperature conventional in the art, preferably 121 °C.
  • the steam sterilization time can be a routine time in the art, preferably 30 minutes.
  • the inoculum amount of the shake flask seed solution may be a conventional inoculum amount in the art, and the volume ratio of the inoculum amount of the shake flask seed liquid to the seed tank seed culture medium is 0.01:1.
  • the temperature of the culture may be a temperature conventional in the art, preferably from 27 to 29 ° C, more preferably 28 ° C.
  • the manner of culturing can be in a manner conventional in the art.
  • the rotation speed of the culture is preferably 200 rpm.
  • the amount of ventilation (air) at the time of the culture is preferably 1 vvm.
  • the culture time may be a conventional culture time in the art, preferably 24 hours.
  • the pH of the obtained seed tank culture solution is generally between 6.8 and 7.0.
  • the concentration of the fibrin-producing fungus hyphae in the seed tank culture solution is generally 25% to 30%, and the percentage refers to the volume of the biodamycin-producing hyphae which accounts for the fermentation of the seed tank. The percentage of the volume of the liquid.
  • the fermentation medium may be a conventional fermentation medium in the art, preferably including sucrose 10 g/L, sorbitol 2 g/L, soluble starch 3 g/L, (NH 4 ) 2 SO 4 0.5 g. /L, beef extract 2g / L, peanut cake powder 1g / L, KH 2 PO 4 0.04g / L, the balance is water, wherein, g / L refers to the mass of each component in the 1L fermentation medium.
  • the fermentation medium may further comprise an antifoaming agent, which may be a conventional antifoaming agent in the art, preferably PPG.
  • the amount of the antifoaming agent used may be a conventional amount in the art, and preferably, it is 1% by mass of the fermentation medium.
  • the fermentation medium is generally sterilized prior to use.
  • the method of sterilization can be a conventional method in the art, preferably steam sterilization.
  • the temperature of the steam sterilization may be a temperature conventional in the art, preferably 121 °C.
  • the steam sterilization time can be a routine time in the art, preferably 20 minutes.
  • the temperature of the culture may be a temperature conventional in the art, preferably from 27 to 29 ° C, more preferably 28 ° C.
  • the manner of culturing can be in a manner conventional in the art.
  • the rotation speed of the culture is preferably from 200 rpm to 300 rpm.
  • the aeration amount at the time of the culture is preferably 0.8 to 1.0 vvm.
  • the culture time may be a conventional culture time in the art, preferably 8 hours.
  • the unit of Fidamycin is generally 2000 mg/L or more, preferably 2800 mg/L to 3200 mg/L.
  • the present invention also provides a fermentation broth containing Fidamycin, which is prepared by cultivating a biological bacteria producing Fidamycin, wherein the Fidamycin-containing fermentation broth is in a unit of More than 2000 mg / L, preferably between 2800 mg / L ⁇ 3200 mg / L.
  • the Fidamycin-producing organism is preferably Actinoplanes sp. HS-16-20; the strain collection number is CGMCC No. 7294.
  • the pretreatment described in the step (a) comprises the steps of: solid-liquid separation of the fermentation liquid containing the Fidamycin to obtain a mycelium, followed by soaking the mycelium with an organic solvent, preferably, the organic solvent It may be a conventional organic solvent for inoculating the mycelium-producing fungus mycelium in the field, preferably methanol or ethanol, more preferably ethanol, and then filtering to obtain a solution containing Fidamycin.
  • the organic solvent may be used in an amount conventionally used in the art.
  • the organic solvent is as described above.
  • the mycelium has a volume-mass ratio of 2.5 to 3.5 L/kg, more preferably 2.9 to 3.0 L/kg (for example, 22 L/7.4 kg, 21 L/7.1 kg, 24 L/8.4 kg, 23 L/7.8 kg).
  • the nanofiltration membrane used in the nanofiltration concentration described in the step (b) may be a conventional nanofiltration membrane in the art, preferably a DK or DL nanofiltration membrane, preferably a DL nanofiltration membrane.
  • the unit of nondamycin in the concentrated solution obtained by concentration after nanofiltration is ⁇ 10000 mg/L.
  • the crude non-damycin can be isolated from the concentrate obtained in the step (b) by a method known in the art.
  • an anti-solvent may be added, preferably water, and the nanofiltration membrane concentrate is added with water so that the volume concentration of the organic solvent in the concentrate (in the concentrate after adding water) is less than 35%, preferably 25%. -30%, the percentage refers to the percentage of the volume of the organic solvent in the concentrate after adding water to the total volume of the concentrate after the addition of water.
  • the nondamycin refined product can be isolated from the eluate obtained in the step (1) by a method known in the art.
  • an anti-solvent preferably water
  • the fermentation pretreatment liquid containing Fidamycin is concentrated by a nanofiltration membrane, and most of the inorganic salts and pigment substances in the fermentation liquid can be removed; finally, the preparation of the C8 filler is selected.
  • the column is such that the impurity and the Fidamycin are effectively separated, and the obtained nondamycin HPLC purity is ⁇ 99.5%, and the yield is ⁇ 50%.
  • the organic solvent used in the present invention can be recycled and reused, and the three wastes are discharged less, and the environment is friendly, which is in line with the current pharmaceutical production trends and requirements.
  • the Actinoplanes sp. HS-16-20 strain used in the present invention has been deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on March 11, 2013, and the deposit address is: Beijing. No. 3, Beichen West Road, Chaoyang District, No. 3, Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101, strain retention number CGMCC NO.7294, classified as Actinoplanes sp., and registered. Prove survival.
  • CGMCC General Microbiology Center
  • FIG. 1 HPLC chromatogram of the Fudamycin fermentation broth in Example 1.
  • the swimming actinomycetes used in the fermentation culture of the present invention have been deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on March 11, 2013, and the preservation number is CGMCC NO.7294; see application date is October 16, 2013.
  • Nanofiltration membrane General Electric Company (GE); Preparation column, Beijing Innovation Tongheng Technology Co., Ltd.; C8 packing, Huapu Xinchuang Technology Co., Ltd., Akzo Nobel; HPLC, Shimadzu LC2010HT;
  • the ethanol and methanol added to the mycelium of the mycelium are commercially available in industrial grade; the methanol and acetonitrile are prepared as commercially available chromatographic grades; and the formic acid is a commercially available reagent grade.
  • the chromatographic purity in the following examples all refers to HPLC purity, and ventilation refers to the introduction of air.
  • the fermentation process of the fermentation broth of the invention is as follows:
  • the plate is made of ISP2 medium, ISP2 medium formula (g/L): glucose 4, yeast extract 4, malt extract 10, agar 15, distilled water to a volume of 1000 mL, pH 7.3, wherein g/L means 1L The mass of each component in the ISP2 medium.
  • the sterilization pressure is 1.05kg/cm 2
  • the sterilization time is 20min
  • the plate is cooled to 50-60°C
  • the cells are connected to the actinomycetes (CGMCC NO.7294). After 8 days of culture at 1 ° C, the hyphae matured and the actinomycetes colonies were obtained.
  • the shake flask volume was 25 mL/250 mL, that is, 25 mL of the seed culture medium was placed in a 250 mL shake flask. The seed medium was sterilized at 121 ° C for 30 minutes before use.
  • the actinomycetes obtained in the step (1) are inoculated into the seed culture medium, the inoculum amount is 10 5 -10 6 cfu/mL, the culture temperature is 28 ⁇ 1 ° C, 250 rpm, and shake culture is carried out for 28 hours.
  • the culture medium has a pH of 6.8-7.0, and the mycelium concentration of the actinomycetes is 25-30% (volume percentage).
  • 100 mL shake flask seed solution was added, the culture temperature was 28 ⁇ 1 ° C, the stirring speed was 200 rpm, the aeration rate was 1 vvm, and the culture was carried out for 24 hours.
  • the culture liquid pH was 6.8-7.0, and the swimming actinomycetes hyphae Concentration 25-30% (v/v).
  • 1% PPG polypropylene glycol
  • the feeding volume is 35L (ie, 35L of fermentation medium), pH 7.0, steam sterilization at 121 ° C for 20 minutes, after cooling, about 3.5L seed tank culture solution is added, the fermentation temperature is 28 ⁇ 1 ° C, and the stirring speed is 200. -300 rpm, aeration of 0.8-1.0 vvm, fermentation culture for 8 days.
  • 30L of non-damycin fermentation unit was 3026mg / L fermentation broth (liquid phase spectrum see Figure 1) filtered to obtain 7.4kg mycelium, the mycelium was put into 22L ethanol soaked, filtered, soaked filtrate, with DK
  • the nanofiltration membrane was concentrated by filtration to a non-damycin unit of more than 10000 mg/L, and purified water was added to the concentrate to an ethanol concentration of 30%. Stirring was continued for 30 min, and the crude nondamycin was filtered (see Figure 2 for the liquid phase map). ), the chromatographic purity was 72.6%.
  • the mobile phase of (V: V: V) was purified by the above crude product, and the fraction with HPLC purity ⁇ 99.5% was collected, and the fraction with purity ⁇ 99.5% was combined. 1.5 volumes of purified water was added under stirring, filtered and dried to obtain Fidamycin.
  • the dry powder was 47.7 g, the chromatographic purity was 99.67% (see Figure 3 for the liquid phase spectrum), and the total extraction yield was 52.5%.
  • the % fraction was added with 1.5 volumes of purified water under stirring, filtered and dried to obtain 45.6 g of dry powder of Fidamycin.
  • the chromatographic purity was 99.58%, and the total extraction yield was 51.3%.
  • the fermentation broth of 30L non-damycin fermentation unit was 3079mg/L, and 8.1kg mycelium was obtained.
  • the mycelium was put into 24L ethanol and soaked, filtered, and the filtrate was collected and concentrated by DL type nanofiltration membrane.
  • the unit of damycin was more than 10000 mg/L, and purified water was added to the concentrate to a methanol concentration of 30%, stirring was continued for 30 min, and crude crude drug was obtained by filtration, and the chromatographic purity was 72.0%.
  • the fraction was added with 1.5 volumes of purified water under stirring, filtered and dried to obtain 48.1 g of dry powder of Fidamycin, the chromatographic purity was 99.62%, and the total extraction yield was 52.1%.
  • the fraction was added with 1.5 volumes of purified water under stirring, filtered and dried to obtain 49.0 g of a dry powder of Fidamycin, the chromatographic purity was 99.68%, and the total extraction yield was 52.7%.

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Abstract

一种纯化非达霉素的方法，由游动放线菌（ Actinoplanes sp.）HS-16-20发酵产生发酵液，所述发酵液经固液分离、有机溶剂浸泡菌丝体、过滤，得到含非达霉素的溶液；所述溶液进行纳滤浓缩、继而分离获得非达霉素粗品；该非达霉素粗品进行制备柱层析，用含有机溶剂的酸水溶液洗脱并分离获得非达霉素精制品。

Description

一种纯化非达霉素的方法
本申请要求申请日为2014年7月9日的中国专利申请CN201410324018.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于医药技术领域，具体涉及抗生素非达霉素的纯化方法。
背景技术
非达霉素(fidaxomicin)是近年来由Op-timer制药公司研发的一种新型窄谱的大环内酯类抗菌药。于2011年5月27日获得FDA批准在美国上市,商品名为Dificid。该药物主要用于治疗艰难梭菌(Clostridium difficile,又名难辨梭状芽孢杆菌)相关性腹泻(CDAD)。
非达霉素的结构式如下：
Figure PCTCN2015083435-appb-000001
目前公开的非达霉素的分离纯化方法主要有：
CN103275152A公开了一种高纯度非达霉素的制备方法，该方法将非达霉素发酵液过滤得到菌丝体，然后将菌丝体用极性溶剂浸泡并固液分离得到非达霉素浸提液，浸提液加水稀释后导入大孔脱色树脂处理得脱色液，脱色液导入大孔吸附树脂吸附饱和后用解析剂梯度洗脱，浓缩，萃取，干燥后得 非达霉素粗品，粗品用极性溶剂溶解后注入聚合物微球柱层析，用洗脱剂进行梯度洗脱，收集洗脱液，合并HPLC测定非达霉素含量大于95％的洗脱液，浓缩、干燥后得到HPLC含量可达97.1％的非达霉素。
CN101663312A公开了一种非达霉素制备方法，该方法在发酵液培养基中加入了吸收剂树脂，在发酵完成后，从发酵液中分离固体物质(包括吸收剂树脂)，用有机溶剂如乙酸乙酯洗脱固体物质，然后减压浓缩得到非达霉素粗品。使用包含Biotage KP-C 18-HS二氧化硅柱纯化，然后浓缩、结晶、干燥得到纯度78％-94.7％的非达霉素。
CN102993251A公开了一种高效液相色谱纯化非达霉素的方法，该方法将纯度为78％的非达霉素粗品经过两次Uni30BPC纯化，得到纯度达到98％的非达霉素洗脱液。
WO2011146621A2公开了一种非达霉素制备方法，该方法用液相制备得到纯度为93％左右的非达霉素。
以上方法制备得到的非达霉素纯度都在99％以下，仍然无法满足药品生产要求，因此急需一种高纯度的非达霉素的制备方法，使其符合药品生产要求。
发明内容
本发明的目的就是提供一种环境友好，工艺简单的纯化非达霉素的方法，本发明的目的可以通过以下技术方案来实现：
一种纯化非达霉素的方法，所述方法包括如下步骤：
(1)将非达霉素粗品进行制备柱层析，用含有有机溶剂的酸水溶液进行洗脱，收集并合并含有非达霉素的洗脱液；和
(2)从步骤(1)所得洗脱液中分离得到非达霉素精制品。
其中，步骤(1)所述的制备柱可为本领域常规的制备柱，一般为反相色谱柱，本发明优选北京创新通恒科技有限公司生产的型号为DAC200制备柱 (200×250mm)。所述的制备柱的填料可为本领域常规的填料，较佳地为C8填料，所述的C8填料是指辛烷基硅烷键合硅胶，一般为本领域常规市售可得，其粒径一般为10μm，可选用华谱C8填料、纳微C8填料、艾杰尔C8填料或kromasilC8填料，优选为kromasilC8填料。
其中，步骤(1)所述的有机溶剂可为本领域常规的有机溶剂，较佳地为甲醇或乙腈；所述酸水溶液为甲酸的水溶液。优选的，步骤(1)所述的含有有机溶剂的酸水溶液为甲醇：水：甲酸＝55-75：45-25：0.1(V:V:V)，或乙腈：水：甲酸＝45-65：55-35：0.1(V:V:V)的溶液，更优选为甲醇：水：甲酸＝60-70：40-30：0.1(V:V:V)，或乙腈：水：甲酸＝50-60：50-40：0.1(V:V:V)的溶液，最优选甲醇：水：甲酸＝65：35：0.1(V:V:V)，或乙腈：水：甲酸＝55：45：0.1(V:V:V)的溶液。以上V:V:V是指体积比。
所述的制备柱层析的方法中，其他色谱条件可为本领域常规的色谱条件，本发明优选下列色谱条件：高效液相色谱仪可为本领域常规的色谱仪，较佳地为津岛公司型号为LC2010HT的高效液相色谱仪。柱温可为本领域常规的柱温，较佳地为20～30℃。检测波长一般为250nm。流速一般为1.0mL/min。进样体积一般为10uL。
其中，步骤(1)中，收集并合并非达霉素HPLC纯度≥99.5％的洗脱液。
其中，步骤(1)所述非达霉素粗品可按照本领域常规的制备方法制备得到，一般要求非达霉素粗品的HPLC纯度在70％以上，例如72.6％、70.9％、72％或73.5％，即可。本发明中，所述的非达霉素粗品是由以下方法制备得到的：
(a)将含有非达霉素的发酵液进行预处理，得到含有非达霉素的溶液；
(b)将步骤(a)所得非达霉素的溶液进行纳滤浓缩，得到含有非达霉素的浓缩液；
(c)从步骤(b)所得浓缩液中分离得到非达霉素粗品。
其中，步骤(a)中，所述的含有非达霉素的发酵液可按照本领域常规的 制备方法制备得到。本发明优选包括下列方法：
①将产非达霉素的生物菌菌体接种至平板培养基中培养，使菌丝成熟，得产非达霉素的生物菌菌落；
②将步骤①得到的产非达霉素的生物菌接种至摇瓶种子培养基中培养，得摇瓶种子液；
③将步骤②得到的摇瓶种子液接种至种子罐种子培养基中培养，得种子罐培养液；
④将步骤③制得的种子罐培养液接种至发酵培养基中培养，即得含有非达霉素的发酵液。
步骤①中，所述的产非达霉素的生物菌一般是指经发酵培养后能够产生非达霉素的生物菌，本发明优选游动放线菌(Actinoplanes sp.)HS-16-20，其已于2013年3月11日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院，中国科学院微生物研究所，菌株保藏编号为CGMCC NO.7294，分类命名为游动放线菌(Actinoplanes sp.)，并登记入册，证明存活。所述的平板培养基可为本领域常规的平板培养基，较佳地为ISP2培养基。所述的ISP2培养基较佳地包括葡萄糖4g/L、酵母提取物4g/L，麦芽抽提物10g/L、琼脂15g/L，其余量为水，其中，g/L是指1L培养基各组分的质量。所述的ISP2培养基中的pH值较佳地为7.3。所述的平板培养基在使用前一般经过灭菌处理。所述的灭菌的方法可为本领域常规的方法。所述的灭菌的压力较佳地为1.05kg/cm2。所述的灭菌的时间较佳地为20min。所述的培养的温度可为本领域常规的温度，较佳地为27～29℃，更佳地为28℃。所述的培养的时间可为本领域常规的时间，只要能够保证产非达霉素的生物菌菌丝成熟即可，较佳地为8天。
步骤②中，所述的摇瓶种子培养基可为本领域常规的摇瓶种子培养基，较佳地，包括蔗糖2g/L、山梨醇3g/L、棉籽饼粉3g/L，花生饼粉1.5g/L，CaCO30.6g/L，MgSO4·7H2O 0.3g/L，其余量为水，其中，g/L是指1L种子 培养基中各组分的质量。所述的摇瓶种子培养基的pH值较佳地为7.2。所述的摇瓶种子培养基在使用前一般经过灭菌处理。所述的灭菌的方法可为本领域常规的方法。所述的灭菌的温度较佳地为121℃。所述的灭菌的时间较佳地为30min。所述的产非达霉素的生物菌菌落的接种量可为本领域常规的接种量，较佳地为每毫升(mL)摇瓶种子培养基中产非达霉素的生物菌的接种量为105～106c.f.u。所述的培养的温度可为本领域常规的温度，较佳地为27～29℃，更佳地为28℃。所述的培养的方式可为本领域常规的方式，较佳地为摇床振荡培养。所述的培养的转速较佳地为250rpm。所述的培养的时间可为本领域常规的培养时间，较佳地为28小时。培养结束后，得到的摇瓶种子液的pH值一般在6.8～7.0之间。所述的摇瓶种子液中的产非达霉素的生物菌菌丝浓度一般在25％～30％，所述的百分比是指产非达霉素的生物菌菌丝的体积占摇瓶种子液的体积的百分比。
步骤③中，所述的种子罐种子培养基可为本领域常规的种子罐种子培养基，较佳地包括蔗糖10g/L，山梨醇2g/L，可溶性淀粉3g/L，(NH4)2SO40.5g/L，牛肉膏2g/L，花生饼粉1g/L，KH2PO40.04g/L，其余量为水，其中，g/L是指1L种子罐培养基中各组分的质量。所述的种子罐种子培养基在使用前一般经过灭菌处理。所述的灭菌的方法可为本领域常规的方法，较佳地为蒸汽灭菌。所述的蒸汽灭菌的温度可为本领域常规的温度，较佳地为121℃。所述的蒸汽灭菌的时间可为本领域常规的时间，较佳地为30min。所述的摇瓶种子液的接种量可为本领域常规的接种量，所述的摇瓶种子液的接种量与所述的种子罐种子培养基的体积比为0.01:1。所述的培养的温度可为本领域常规的温度，较佳地为27～29℃，更佳地为28℃。所述的培养的方式可为本领域常规的方式。所述的培养的转速较佳地为200rpm。所述的培养时的通气(空气)量较佳地为1vvm。所述的培养的时间可为本领域常规的培养时间，较佳地为24小时。培养结束后，得到的种子罐培养液的pH值一般在6.8～7.0之间。所述的种子罐培养液中的产非达霉素的生物菌菌丝浓度一般在 25％～30％，所述的百分比是指产非达霉素的生物菌菌丝的体积占种子罐发酵液的体积的百分比。
步骤④中，所述的发酵培养基可为本领域常规的发酵培养基，较佳地包括蔗糖10g/L，山梨醇2g/L，可溶性淀粉3g/L，(NH4)2SO40.5g/L，牛肉膏2g/L，花生饼粉1g/L，KH2PO40.04g/L，其余量为水，其中，g/L是指1L发酵培养基中各组分的质量。所述的发酵培养基中还可进一步包含消泡剂，所述的消泡剂可为本领域常规的消泡剂，较佳地为PPG。所述的消泡剂的用量可为本领域常规的用量，较佳地，其为发酵培养基质量的1％。所述的发酵培养基在使用前一般经过灭菌处理。所述的灭菌的方法可为本领域常规的方法，较佳地为蒸汽灭菌。所述的蒸汽灭菌的温度可为本领域常规的温度，较佳地为121℃。所述的蒸汽灭菌的时间可为本领域常规的时间，较佳地为20min。所述的培养的温度可为本领域常规的温度，较佳地为27～29℃，更佳地为28℃。所述的培养的方式可为本领域常规的方式。所述的培养的转速较佳地为200rpm～300rpm。所述的培养时的通气量较佳地为0.8～1.0vvm。所述的培养的时间可为本领域常规的培养时间，较佳地为8小时。
按照上述制备方法得到的含非达霉素的发酵液中，非达霉素的单位一般在2000mg/L以上，较佳地为2800mg/L～3200mg/L之间。
本发明还提供了一种含有非达霉素的发酵液，其通过培养产非达霉素的生物菌制得，所述的含有非达霉素的发酵液中，非达霉素的单位在2000mg/L以上，较佳地为2800mg/L～3200mg/L之间。所述的产非达霉素的生物菌优选游动放线菌(Actinoplanes sp.)HS-16-20；菌株保藏编号为CGMCC NO.7294。
其中，步骤(a)所述的预处理包括以下步骤：将含有非达霉素的发酵液进行固液分离得到菌丝体，接着将菌丝体用有机溶剂浸泡，优选的，所述有机溶剂可为本领域浸泡产非达霉素的生物菌菌丝体常规的有机溶剂，较佳地为甲醇或者乙醇，更优选为乙醇，然后过滤得到含有非达霉素的溶液。所述的有机溶剂的用量可为本领域常规的用量，较佳地，所述的有机溶剂与所述 的菌丝体的体积质量比为2.5～3.5L/kg，更佳地为2.9～3.0L/kg(例如22L/7.4kg、21L/7.1kg、24L/8.4kg、23L/7.8kg)。
其中，步骤(b)所述的纳滤浓缩使用的纳滤膜可为本领域常规的纳滤膜，较佳地为DK或DL纳滤膜，优选为DL纳滤膜。优选的，经过纳滤浓缩后得到的浓缩液中非达霉素的单位≥10000mg/L。
其中，可采用本领域公知的方法从步骤(b)所得浓缩液中分离得到非达霉素粗品。在一个具体的实施例中，可以加入一种反溶剂，优选水，纳滤膜浓缩液加水后使得浓缩液中(加入水后的浓缩液中)有机溶剂的体积浓度小于35％，优选25％-30％，所述的百分比是指加入水后的浓缩液中，有机溶剂的体积占加入水后的浓缩液的总体积的百分比。
其中，可采用本领域公知的方法从步骤(1)所得洗脱液中分离得到非达霉素精制品。在一个具体的实施例中，可以加入一种反溶剂，优选水，水的加入量与步骤(1)收集到的洗脱液的体积比为1-2:1，优选1.4-1.6:1。
本发明的优势在于：
本发明在非达霉素粗品的制备过程中，将含有非达霉素的发酵预处理液采用纳滤膜浓缩，可以去除发酵液中大部分无机盐和色素类物质；最后选用C8填料的制备柱，使得杂质和非达霉素得到有效分离，最后得到的非达霉素HPLC纯度≥99.5％，收率≥50％。另外，本发明所用有机溶剂可回收重复使用，三废排放少，环境友好，非常符合当今医药生产趋势和要求。
生物材料保藏信息
本发明所采用的游动放线菌(Actinoplanes sp.)HS-16-20菌株，已于2013年3月11日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编：100101，菌株保藏编号为CGMCC NO.7294，分类命名为游动放线菌(Actinoplanes sp.)，并登记入册，证明存活。
附图说明
图1实施例1中的非达霉素发酵液HPLC图谱
图2实施例1制备得到的非达霉素粗品沉淀物HPLC图谱
图3实施例1制备得到的非达霉素精制品HPLC图谱
具体实施方式
本发明发酵培养所用游动放线菌已于2013年3月11日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.7294；具体见申请日为2013年10月16日的中国专利申请CN201310501389.3，在此本申请引用上述中国专利申请的全文。纳滤膜，通用电气公司(GE)；制备柱，北京创新通恒科技有限公司；C8填料，华谱新创科技有限公司、阿克苏诺贝尔公司；高效液相色谱仪，岛津LC2010HT；非达霉素菌丝体中加入的乙醇、甲醇为市售工业级；制备用甲醇、乙腈为市售色谱级；甲酸为市售试剂级。下述实施例中的色谱纯度均是指HPLC纯度，通气是指通入空气。
本发明发酵液培养过程如下：
(1)平板菌落的制备与培养
平板采用ISP2培养基，ISP2培养基配方(g/L)：葡萄糖4，酵母抽提物4，麦芽抽提物10，琼脂15，蒸馏水定容至1000mL，pH 7.3，其中，g/L是指1L ISP2培养基中各组分的质量。在使用前经过灭菌处理，灭菌压力1.05kg/cm2，灭菌时间20min，冷却到50-60℃倒平板，接入游动放线菌(CGMCC NO.7294)菌体，经28±1℃培养8天后，菌丝成熟，得游动放线菌菌落。
(2)摇瓶种子液的制备与培养
种子培养基配方(g/L)：蔗糖2，山梨醇3，棉籽饼粉3，花生饼粉1.5，CaCO30.6，MgSO4·7H2O 0.3，pH 7.2，其余量为水，其中，g/L是指1L种子 培养基中各组分的质量。摇瓶装液量为25mL/250mL，即250mL摇瓶中装入25mL种子培养基。种子培养基在使用前经121℃灭菌30分钟。然后将步骤(1)得到的游动放线菌接种至种子培养基中，接种量为105-106c.f.u./mL，培养温度28±1℃，250rpm，摇床振荡培养28小时，此时培养液pH 6.8-7.0，(游动放线菌)菌丝浓度25-30％(体积百分比)。
(3)种子罐培养液的制备
在15L种子罐中投入10L的种子培养基(种子罐培养基配方(g/L)：蔗糖10，山梨醇2，可溶性淀粉3，(NH4)2SO40.5，牛肉膏2，花生饼粉1，KH2PO40.04，其余量为水，其中，g/L是指1L种子罐培养基中各组分的质量)，灭菌采用蒸汽灭菌，121℃条件下30分钟。待冷却后，接入100mL摇瓶种子液，培养温度为28±1℃，搅拌转速200rpm，通气量1vvm，培养24小时，此时培养液pH 6.8-7.0，(游动放线菌)菌丝浓度25-30％(v/v)。
(4)发酵罐培养基的配制与培养
发酵培养基配方(g/L)：蔗糖10，山梨醇2，可溶性淀粉3，(NH4)2SO40.5，牛肉膏2，花生饼粉1，KH2PO40.04，其余量为水，其中，g/L是指1L发酵培养基中各组分的质量。所述的发酵培养基中添加1％PPG(聚丙二醇)作为消泡剂。投料体积为35L(即发酵培养基35L)，pH7.0，于121℃条件下蒸汽灭菌20分钟，冷却后，接入约3.5L种子罐培养液，发酵温度28±1℃，搅拌转速200-300rpm，通气量0.8-1.0vvm，发酵培养8天。
实施例1
将30L非达霉素发酵单位为3026mg/L的发酵液(液相图谱见附图1)过滤后得到7.4kg菌丝体，将菌丝体投入22L乙醇浸泡后过滤，收集浸泡滤液，用DK型纳滤膜纳滤浓缩至非达霉素单位大于10000mg/L，向浓缩液加入纯化水至乙醇浓度为30％，继续搅拌30min，过滤得非达霉素粗品(液相图谱见附图2)，色谱纯度为72.6％。
使用装有华谱C8填料的DAC200制备柱，乙腈：水：甲酸＝55：45：0.1 (V：V：V)的流动相纯化上述粗品，收集HPLC纯度≥99.5％的馏分，合并纯度≥99.5％的馏分，搅拌下加入1.5倍体积的纯化水，过滤、干燥，得到非达霉素干粉47.7g，色谱纯度99.67％(液相图谱见附图3)，提取总收率52.5％。
实施例2
将30L非达霉素发酵单位为2963mg/L的发酵液过滤后得到7.1kg菌丝体，将菌丝体投入21L甲醇浸泡后过滤，收集浸泡滤液，用DK型纳滤膜纳滤浓缩至非达霉素单位大于10000mg/L，向浓缩液加入纯化水至甲醇浓度为30％，继续搅拌30min，过滤得非达霉素粗品，色谱纯度为70.9％。
使用装有华谱C8填料的DAC200制备柱，甲醇：水：甲酸＝65：35：0.1(V：V：V)的流动相纯化上述粗品，收集HPLC纯度≥99.5％的馏分，合并纯度≥99.5％的馏分，搅拌下加入1.5倍体积的纯化水，过滤、干燥，得到非达霉素干粉45.6g，色谱纯度99.58％，提取总收率51.3％.
实施例3
将30L非达霉素发酵单位为3079mg/L的发酵液过滤后得到8.1kg菌丝体，将菌丝体投入24L乙醇浸泡后过滤，收集浸泡滤液，用DL型纳滤膜纳滤浓缩至非达霉素单位大于10000mg/L，向浓缩液加入纯化水至甲醇浓度为30％，继续搅拌30min，过滤得非达霉素粗品，色谱纯度为72.0％。
使用装有kromasilC8填料的DAC200制备柱，乙腈：水：甲酸＝55：45：0.1(V：V：V)的流动相纯化上述粗品，收集HPLC纯度≥99.5％的馏分，合并纯度≥99.5％的馏分，搅拌下加入1.5倍体积的纯化水，过滤、干燥，得到非达霉素干粉48.1g，色谱纯度99.62％，提取总收率52.1％.
实施例4
将30L非达霉素发酵单位为3102mg/L的发酵液过滤后得到7.8kg菌丝体，将菌丝体投入23L乙醇浸泡后过滤，收集浸泡滤液，用DL型纳滤膜纳滤浓缩至非达霉素单位大于10000mg/L，向浓缩液加入纯化水至乙醇浓度为30％，继续搅拌30min，过滤得非达霉素粗品，色谱纯度为73.5％。
使用装有kromasilC8填料的DAC200制备柱，甲醇：水：甲酸＝65：35：0.1(V：V：V)的流动相纯化上述粗品，收集HPLC纯度≥99.5％的馏分，合并纯度≥99.5％的馏分，搅拌下加入1.5倍体积的纯化水，过滤、干燥，得到非达霉素干粉49.0g，色谱纯度99.68％，提取总收率52.7％。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (18)

  1. 一种纯化非达霉素的方法，其特征在于，所述方法包括如下步骤：
    (1)将非达霉素粗品进行制备柱层析，用含有有机溶剂的酸水溶液进行洗脱，收集并合并含有非达霉素的洗脱液；和
    (2)从步骤(1)所得洗脱液中分离得到非达霉素精制品。
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)所述非达霉素粗品是由以下方法制备得到的：
    (a)将含有非达霉素的发酵液进行预处理，得到含有非达霉素的溶液；
    (b)将步骤(a)所得非达霉素的溶液进行纳滤浓缩，得到含有非达霉素的浓缩液；
    (c)从步骤(b)所得浓缩液中分离得到非达霉素粗品。
  3. 根据权利要求2所述的方法，其特征在于，步骤(a)所述预处理包括以下步骤：将含有非达霉素的发酵液进行固液分离得到菌丝体，接着将菌丝体用有机溶剂浸泡，然后过滤得到含有非达霉素的溶液。
  4. 根据权利要求3所述的方法，其特征在于，步骤(a)所述预处理中所述有机溶剂为甲醇或者乙醇。
  5. 根据权利要求2-4中至少一项所述的方法，其特征在于，步骤(a)所述的含有非达霉素的发酵液的制备方法包括下列步骤：
    ①将产非达霉素的生物菌菌体接种至平板培养基中培养，使菌丝成熟，得产非达霉素的生物菌菌落；
    ②将步骤①得到的产非达霉素的生物菌接种至摇瓶种子培养基中培养，得摇瓶种子液；
    ③将步骤②得到的摇瓶种子液接种至种子罐种子培养基中培养，得种子罐培养液；
    ④将步骤③制得的种子罐培养液接种至发酵培养基中培养，即得含有非 达霉素的发酵液；
    其中，所述的产非达霉素的生物菌为游动放线菌(Actinoplanes sp.)HS-16-20，菌株保藏编号为CGMCC NO.7294。
  6. 根据权利要求2-5中至少一项所述的方法，其特征在于，步骤(b)所述的纳滤浓缩使用的纳滤膜为DK或DL纳滤膜。
  7. 根据权利要求2-6中至少一项所述的方法，其特征在于，步骤(b)所述的含有非达霉素的浓缩液中非达霉素的单位≥10000mg/L。
  8. 根据权利要求2-7中至少一项所述的方法，其特征在于，步骤(c)为向步骤(b)所得浓缩液中加入反溶剂，然后分离得到非达霉素粗品。
  9. 根据权利要求8所述的方法，其特征在于，所述的反溶剂为水，所述的水的加入的量使得浓缩液中有机溶剂的体积浓度小于35％，所述的百分比是指加入水后的浓缩液中，有机溶剂的体积占加入水后的浓缩液总体积的百分比。
  10. 根据权利要求1-9中至少一项所述的方法，其特征在于，步骤(1)所述的制备柱填料为C8填料。
  11. 根据权利要求1-10中至少一项所述的方法，其特征在于，步骤(1)所述的有机溶剂为甲醇或乙腈。
  12. 根据权利要求1-11中至少一项所述的方法，其特征在于，步骤(1)所述酸水溶液为甲酸的水溶液。
  13. 根据权利要求1-12中至少一项所述的方法，其特征在于，步骤(1)所述的含有有机溶剂的酸水溶液为甲醇：水：甲酸的体积比＝55-75：45-25：0.1，或乙腈：水：甲酸的体积比＝45-65：55-35：0.1的溶液。
  14. 根据权利要求13所述的方法，其特征在于，步骤(1)所述的含有有机溶剂的酸水溶液为甲醇：水：甲酸的体积比＝60-70：40-30：0.1，或乙腈：水：甲酸的体积比＝50-60：50-40：0.1的溶液。
  15. 根据权利要求1-14中至少一项所述的方法，其特征在于，步骤(1) 中，收集并合并非达霉素HPLC纯度≥99.5％的洗脱液。
  16. 根据权利要求1-15中至少一项所述的方法，其特征在于，向步骤(1)所得洗脱液中加入反溶剂，从步骤(1)所得洗脱液中分离得到非达霉素精制品。
  17. 根据权利要求1-16中至少一项所述的方法，其特征在于，所述的向步骤(1)所得洗脱液中加入的反溶剂为水，所述的水的加入量与步骤(1)收集到的洗脱液的体积比为1-2:1。
  18. 一种含有非达霉素的发酵液，其特征在于，其通过培养产非达霉素的生物菌制得，所述的含有非达霉素的发酵液中，非达霉素的单位在2000mg/L以上；所述的产非达霉素的生物菌为游动放线菌(Actinoplanes sp.)HS-16-20，菌株保藏编号为CGMCC NO.7294。
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