CN101463076B - Method for refining bleocin - Google Patents
Method for refining bleocin Download PDFInfo
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- CN101463076B CN101463076B CN2007101725198A CN200710172519A CN101463076B CN 101463076 B CN101463076 B CN 101463076B CN 2007101725198 A CN2007101725198 A CN 2007101725198A CN 200710172519 A CN200710172519 A CN 200710172519A CN 101463076 B CN101463076 B CN 101463076B
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- bleomycin
- organic solvent
- bleocin
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- purification
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Abstract
The invention discloses a refining method for bleocin, comprising purifying the crude product of bleocin by preparative HPLC technology; wherein, the immobile phase of a preparation column is silica gel column of C8 or C18, the mobile phase is mixed liquid of sulphate aqueous solution with pH value between 3.5 and 5 and polar organic solvent. In the method, the preparative HPLC technology is adopted to increase the yield (85% generally) and the degree of automation of A2 and A3 which are the active ingredients of bleocin and greatly lower cost, thus can be applied to industrial production.
Description
Technical field
The present invention relates to a kind of process for purification of bleomycin.
Background technology
Because the deterioration and the unsound living habit of environment, worldwide tumor incidence continues to increase.The annual tumour patient of newly making a definite diagnosis in the whole world is all more than 1,030 ten thousand people since 1996, to the end of the year 1999 whole world tumour patient sum exceeded 4,000 ten thousand people.World Health Organization's calendar year 2001 report, world's cancer morbidity and mortality ratio have risen 22% than the nineteen ninety, also will rise about 50% in 20 years from now on.
Bleomycin (Bleomycin) is the broad-spectrum antimicrobial antitumor antibiotics that gang has unique texture and effect, and is effective to the kinds of tumors disease, and do not cause and do not suppress body's immunity by oligoleukocythemia, do not damage hemopoietic system.The mechanism of bleomycin killing tumor cell is to combine with iron and oxygen in vivo to form mixture, then with karyomit(e) effect attack DNA target spot, causes the dna degradation reaction, thereby synthetic by suppressing tumour cell DNA, reaches antineoplastic effect.So the exploitation bleomycin has important practical significance.
Bleomycin is a family antibiotic, is made of 5 amino acid, 2 hexoses and 1 amino side-chain, and each component molecular structure differences only is the amino difference that replaces side chain, and the wherein present clinical bleomycin main component of using is A2 and B2 component.Because structure is special and contain more homologue, bleomycin extracts and acquires a certain degree of difficulty.Existing extracting method be through slightly carry obtain the bleomycin crude product after, make with extra care this process for purification yield lower (generally about 70%), and level of automation is not high with gel chromatography.
Summary of the invention
Therefore, the problem to be solved in the present invention just provides a kind of process for purification of bleomycin, and this process for purification yield is higher, and the preparation time spent is shorter, and cost is lower.
The inventor is through discovering: available technology adopting high performance liquid chromatography (HPLC) analytical technology detects bleomycin unit, and this HPLC technology can develop in order to refining bleomycin.But the ion pair reagent that adds in the moving phase of existing HPLC analytical procedure all costs an arm and a leg, as adding sodium pentanesulfonate in the moving phase in the HPLC condition of analyzing bleomycin in the American Pharmacopeia, its price is generally in per 100 gram 2000-3000 units, if so in industrial production, make with extra care bleomycin on a large scale with it, cost is too high, is infeasible.The inventor is through a large amount of tests, and find: alternative this ion pair reagent of several more common and cheap soluble sulphate classes greatly reduces production cost, thereby realized the present invention pleasantly surprisedly.
Therefore, the present invention solves the problems of the technologies described above the technical scheme that is adopted and can be: a kind of process for purification of bleomycin comprises the bleomycin crude product is adopted preparation HPLC technology purifying that wherein the stationary phase of preparative column is C
8Or C
18Silicagel column, moving phase is the sulfate solution of pH3.5～5 and the mixed solution of polar organic solvent.
Vitriol of the present invention generates metal ion or ammonium radical ion (NH during for ionization
4 +), and sulfate ion (SO
4 2-) compound, and be dissolvable in water in the aqueous solution of described pH3.5～5.Preferable described vitriol is optional from vitriolate of tartar, one or more in sodium sulfate and the ammonium sulfate.That the consumption of described vitriol is preferable is 0.05～0.5% (g/100ml) that accounts for the described aqueous solution.
Preferable, the sulfate solution of described pH3.5～5 is to transfer pH to 3.5～5 to form by the aqueous solution that is dissolved with vitriol with acetic acid.
Polar organic solvent of the present invention can be the polar organic solvent of institute's energy usefulness in the existing bleomycin HPLC analytical procedure, preferable acetonitrile and/or the methyl alcohol of can be.
All the other conditions of preparation HPLC technology of the present invention all can be with reference to existing analysis mode HPLC technology, as detect wavelength and can be existing any wavelength that can detect bleomycin, 280～290nm or 250～260nm that preferred analysis mode HPLC technology is often selected for use; Preferred 10～the 20ml/min of flow rate of mobile phase, more preferably 13-16ml/min; Moving phase can adopt isocratic elution, the more preferably method of gradient elution: the ratio of polar organic solvent in moving phase gradually from low to high, behind certain proportion, keep for some time, usually the organic phase ratio is increased to 25～40% in 10～20% (v/v) scope in about 25～35 minutes time, keeps about 5～15 minutes.
Bleomycin crude product of the present invention for the bleomycin fermented liquid by prior art for preparing through preliminary purification but not further as the bleomycin sample (HamaoUmezawa.Purification of bleomycins.The journal of antibiotics.Sept.1966p210-215) that adopts the refining purifying of gel column to obtain.
The process for purification of bleomycin of the present invention has following advantage than prior art:
1) the present invention adopts preparation HPLC technology can obtain higher yield (generally 85%), and the gel chromatography process for purification yield of existing bleomycin is generally 70%;
2) the present invention adopts preparation HPLC technology, has improved level of automation;
3) as ion pair reagent, the vitriol price about about 10 yuan of per 500 grams, greatly reduces cost greatly, can be used for suitability for industrialized production with several more common and cheap soluble sulphates in the present invention.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
The instrument that uses in the following example prepares liquid phase as the special P270 type of Dalian Erie.The experimental technique of unreceipted actual conditions, usually according to normal condition, or the condition of advising according to manufacturer.
Wherein the calculation formula of yield (%) is: (A2+B2) component * 100% of (A2+B2) component of collecting/when going up sample.
Embodiment 1
Use C
18(Hypersil filler, 20mm * 250mm 10um), detect wavelength 254nm to silicagel column.The bleomycin crude product is with water dissolution (200mg is dissolved in the 10ml water, each sample introduction 1ml).Proportion of mobile phase: ammonium sulfate 1.0 grams, be dissolved in the 1000ml water, transfer pH to 3.5 with acetic acid, be organic phase with methyl alcohol.Flow velocity 15ml/min, the organic phase ratio rises to 30% by 15% in 30min, and keeps 10min, and each component of bleomycin can be separated well, and yield is 87.2%.
Embodiment 2
Use C
8(Hypersil filler, 20mm * 250mm 10um), detect wavelength 254nm to silicagel column.The bleomycin crude product is with water dissolution (100mg is dissolved in the 10ml water, each sample introduction 2ml).Proportion of mobile phase: vitriolate of tartar 5.0 grams, be dissolved in the 1000ml water, transfer pH to 4.5 with acetic acid, be organic phase with the acetonitrile.Flow velocity 16ml/min, the organic phase ratio rises to 28% by 12% in 30min, and keeps 10min, and each component of bleomycin can be separated well, yield 88.7%.
Embodiment 3
Use C
18(Hypersil filler, 20mm * 250mm 10um), detect wavelength 280nm to silicagel column.The bleomycin crude product is with water dissolution (150mg is dissolved in the 10ml water, each sample introduction 1ml).Proportion of mobile phase: sodium sulfate 0.5 gram, be dissolved in the 1000ml water, transfer pH to 5 with acetic acid, be organic phase with methyl alcohol.Flow velocity 13ml/min, the organic phase ratio rises to 35% by 1 8% in 35min, and keeps 10min, and each component of bleomycin can be separated well, and yield is 85.3%.
Claims (4)
1. the process for purification of a bleomycin, it comprises the bleomycin crude product is adopted preparation HPLC technology purifying that wherein the stationary phase of preparative column is C
8Or C
18Silicagel column, moving phase is the sulfate solution of pH3.5～5 and the mixed solution of polar organic solvent, described polar organic solvent is acetonitrile and/or methyl alcohol, flow rate of mobile phase 10～20ml/min, moving phase adopts gradient elution: in 25～35 minutes in the moving phase organic solvent ratio in 10～20v/v% scope, be increased to 25～40v/v%, kept 5～15 minutes.
2. process for purification according to claim 1 is characterized in that described vitriol is selected from vitriolate of tartar, sodium sulfate and ammonium sulfate.
3. process for purification according to claim 1 is characterized in that, the content of described vitriol in the aqueous solution is 0.05～0.5w/v%.
4. process for purification according to claim 1 is characterized in that, the sulfate solution of described pH3.5～5 is to transfer pH to 3.5～5 to form by the aqueous solution that is dissolved with vitriol with acetic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101725198A CN101463076B (en) | 2007-12-18 | 2007-12-18 | Method for refining bleocin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101725198A CN101463076B (en) | 2007-12-18 | 2007-12-18 | Method for refining bleocin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101463076A CN101463076A (en) | 2009-06-24 |
| CN101463076B true CN101463076B (en) | 2011-08-03 |
Family
ID=40803894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007101725198A Expired - Fee Related CN101463076B (en) | 2007-12-18 | 2007-12-18 | Method for refining bleocin |
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| CN (1) | CN101463076B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004065B (en) * | 2013-02-26 | 2017-10-20 | 长沙天赐生物医药科技有限公司 | A kind of Isolation and purification method of bleomycin race derivative |
| CN104231057B (en) * | 2013-06-21 | 2017-06-20 | 哈尔滨莱博通药业有限公司 | The purification process of the copper chelate of bleomycin A5 and its congeners |
| CN104877010B (en) * | 2015-06-15 | 2018-05-29 | 华北制药集团新药研究开发有限责任公司 | A kind of preparation method of pristinamycin I A one pack systems |
-
2007
- 2007-12-18 CN CN2007101725198A patent/CN101463076B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| A. Aszalos et al.High-performance liquid chromatographic determination of components of bleomycin preparations.《journal of pharmaceutical sciences》.1981,第70卷(第8期),878-880. * |
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| CN101463076A (en) | 2009-06-24 |
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