CN101367855B - Method for purification of erythromycin A - Google Patents
Method for purification of erythromycin A Download PDFInfo
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- CN101367855B CN101367855B CN2008102003780A CN200810200378A CN101367855B CN 101367855 B CN101367855 B CN 101367855B CN 2008102003780 A CN2008102003780 A CN 2008102003780A CN 200810200378 A CN200810200378 A CN 200810200378A CN 101367855 B CN101367855 B CN 101367855B
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- erythromycin
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Abstract
The present invention relates to a method for purifying erythromycin A from erythromycin base (crude product). The purifying method mainly includes the following steps: the erythromycin base undergoes column chromatography to produce target compound, which is characterized in that the adsorption resin used for column chromatography is polystyrene type nonpolar macroporous adsorption resin; the eluent used for column chromatography is one or two or more selected from the mixed solution of C1-C6 monohydric fatty alcohol and water, the mixed solution of C2-C6 fatty ether and water, the mixed solution of C2-C6 fatty ester and water or the mixed solution of C3-C6 fatty ketone and water. Compared with the prior art, the method has the advantages of simple operation, economy, high product purity and yield, and is suitable for mass commercial preparation.
Description
Technical field
The present invention relates to a kind of method of purification of erythromycin A, particularly a kind of Erythromycin A purification process of from crude product erythromycin (its main ingredient is Erythromycin A and Erythromycin C), removing highly toxic Erythromycin C, obtaining to satisfy medical standard Erythromycin A.
Background technology
Erythromycin A is a kind of in the many isomer of erythromycin, because of it has hypotoxicity and high germ resistance, so become the main anti-microbial activity composition of medical erythromycin.Usually, the erythromycin (crude product) that makes through fermentation is for comprising the multiple mixture of isomers of erythromycin, and therefore, the separation and purification of one-component receives much attention in the erythromycin (crude product).
So far, the method that is used for separation and purification erythromycin (crude product) has: solvent extration, membrane separation process (Chinese JournalofAntibiotics, 2003,28 (10): 597-604; Biotechnology and Bioengineering, 2003,81 (6): 640-649), ion exchange method, macroreticular resin absorbing method (Chinese microbiotic magazine, 1991,16 (4): 266-270; Bioprocess BiosystEng, 2003,26:49-55.) and high speed adverse current chromatogram preparation method (CN101104631) etc.
Shortcoming such as wherein there is poor selectivity in solvent extration, solvent load is big and the rate of recovery is low; Membrane separation technique exists the easily contaminated and cost of product than problems such as height; Existing ion exchange method and macroporous adsorbent resin method are from fermented liquid or stock liquid extracts erythromycin, does not relate to the Separation Research of erythromycin component as yet; Though the high speed adverse current chromatogram preparation method is the one-component (as Erythromycin A, berythromycin or Erythromycin C) of (yield of target compound and purity are all comparatively desirable) acquisition erythromycin better, this method is not suitable for the mass-producing commercial production.
Given this, the separation purification method of one-component that a kind of product purity and yield height is provided and is suitable for the erythromycin of mass-producing commercial production just becomes the technical issues that need to address of the present invention.
Summary of the invention
The objective of the invention is to, provide a kind of and from crude product erythromycin (its main ingredient is Erythromycin A and Erythromycin C), remove highly toxic Erythromycin C, obtain to satisfy medical standard Erythromycin A and be suitable for the Erythromycin A purification process of mass-producing commercial production.
The method of the said purification of erythromycin A of the present invention, its key step is: with crude product erythromycin (its contained major impurity is an Erythromycin C, the about 8wt% of content) is raw material, and erythromycin (crude product) is obtained target compound behind column chromatography, it is characterized in that,
The polymeric adsorbent that is used for said column chromatography is the polystyrene type nonpolar macroporous adsorption resin;
The eluent that is used for said column chromatography is selected from: C
1～C
6The monobasic fatty alcohol and mixed solution, the C of water
2～C
6Fatty ether and mixed solution, the C of water
2～C
6The mixed solution of aliphatic ester and water or C
3～C
6A kind of in the mixed solution of aliphatic ketone and water, two or more;
Said column chromatography is to carry out under 0 ℃～55 ℃ the condition in temperature.
In the present invention, raw materials used (crude product erythromycin) by: fermentation method prepares the fermented liquid of erythromycin gained and (comprises steps such as filtration, membrane sepn, extraction and precipitation through the initial gross separation of prior art, see for details: print 2007,21 (1): obtain 59-62.) during chemical industry.
In optimal technical scheme of the present invention, the polymeric adsorbent that is used for said column chromatography is that the aperture is greater than 40
, surface-area is greater than 400m
2The polystyrene type nonpolar macroporous adsorption resin of/g.Amberlite XAD-16, Amberlite XAD-1600, Amberlite XAD-1180, SEPABEADS SP-825, the SEPABEADS SP-850 of Mitsubishi, DIAION HP-20, DIAION HP-21 etc. as (but being not limited to): the HZ of Shanghai Huazhen Science and Technology Co., Ltd.-816, HZ-818, HZ-820, HZ-826, U.S. Rohm-hass company.
In another optimal technical scheme of the present invention, the eluent that is used for said column chromatography is C
1～C
6Unitary fatty alcohol and the mixed solution or the C of water
2～C
6Fatty ester and the mixed solution of water; Preferred eluent is C
1～C
3The aqueous solution or the C of unitary fatty alcohol
4～C
6The aqueous solution of fatty ester; Best eluent is that concentration is 1v/v% aqueous ethanolic solution～100v/v% ethanol, the 1v/v%～7v/v% vinyl acetic monomer aqueous solution or vinyl acetic monomer.
In another optimal technical scheme of the present invention, the add-on (by weight) of raw material [erythromycin (crude product)] is 0.2～0.8 times of saturated extent of adsorption (by weight) of selected polymeric adsorbent.
In another optimal technical scheme of the present invention,, the some identical chromatography column series connection of separation performance can be used for improving the yield of product (Erythromycin A).Under comprehensive consideration product yield, separation efficiency and preparation cost prerequisite, the present invention recommends to adopt two identical chromatography column series connection of separation performance to use.
Embodiment
The method of the said purification of erythromycin A of the present invention comprises the steps:
(1) with raw material [erythromycin (crude product), it consists of: the Erythromycin A of 87.2wt%, the Erythromycin C of 7.6wt% and other impurity of 5.2wt%] and C
1～C
3The aqueous solution of unitary fatty alcohol get stock liquid, the pH value of regulating the gained stock liquid is 6.0～9.0, break into portions is standby;
(2) macroporous adsorbent resin SEPABEADS SP835 (the Mitsubishi chemistry is produced) is filled in two diameters and the identical length chromatography column together (loadings of macroporous adsorbent resin is also identical), and it is connected in series, with C
1～C
3The solution washing of unitary fatty alcohol to being connected in series with two UV-detector baseline balances behind the chromatography column;
To be that 6.0～9.0 stock liquid joins with in one in two chromatography columns of series system connection by a pH value of step (1) gained, be easy narration, we will be added with the pH value is that the chromatography column of 6.0～9.0 stock liquid is defined as " main chromatography column ", corresponding, another root then is called " auxilliary chromatography column "; Wherein: the pH value is that the amount (by weight) that 6.0～9.0 stock liquid is added in " main chromatography column " is 0.2～0.8 times of " main chromatography column " saturated extent of adsorption (by weight);
C with 5v/v%
1～C
3The aqueous solution of unitary fatty alcohol be eluent a, respectively " main chromatography column " and " auxilliary chromatography column " carried out wash-out with eluent a.
Adopt the concentration of the Erythromycin A in high performance liquid chromatography (HPLC) monitoring " main chromatography column " effluent liquid, when the concentration of Erythromycin A in " main chromatography column " effluent liquid greater than 90% the time, change eluent.Promptly with the C of 95v/v%
1～C
3The aqueous solution of unitary fatty alcohol be eluent b, with eluent b " main chromatography column " carried out wash-out, collect elutriant, after drying target compound (Erythromycin A).
(3) " main chromatography column " in the step (2) regenerated and balance [said renovation process is asked for an interview: Heilungkiang medical science, 2007,31 (3): 237-238; The same step of balance method (2)] after connect with " auxilliary chromatography column " in the step (2) again, carry out then " blocked operation ", that is: be that 6.0～9.0 mixed solution adds in the step (2) " auxilliary chromatography column " (according to the definition of preamble by step (1) another part of gained pH value, it is existing to be " main chromatography column "), repeating step (2) gets target compound.
So, " main chromatography column " and " auxilliary chromatography column " carried out " blocked operation ", and " blocked operation " is preceding each time, last round-robin " main chromatography column " must be reproduced and balance.
In with eluent a elution process, when beginning to contain Erythromycin A in " in case auxilliary chromatography column " effluent liquid, promptly finish blocked operation, and " auxilliary chromatography column " carried out wash-out with eluent b, collect elutriant, through concentrated, crystallization (East China University of Science's journal (natural science edition), 2006,32 (8): 897-901; China's microbiotic magazine, 1998,23 (1): 14-16; External medical microbiotic fascicle, 2001,22 (5): 207-210.) etc. method reclaims Erythromycin A, " auxilliary chromatography column " is regenerated, come into operation after the balance, begins twin columns once more and switches cyclical operation.
The invention provides a kind of (sorbent material and eluent consumption are few) simple to operate, economical and yield higher, by removing the Erythromycin A purification process that highly toxic Erythromycin C, acquisition satisfy medical standard Erythromycin A in the crude product erythromycin (its main ingredient is Erythromycin A and Erythromycin C).Compare with the method for existing separation and purification Erythromycin A, that the present invention not only has is simple to operate, economy and yield advantages of higher and be easy to the mass-producing commercial production.
The present invention is further elaborated below by embodiment, and its purpose only is better to understand content of the present invention.Therefore, for embodiment do not limit protection scope of the present invention:
Embodiment 1
Compound concentration is the 5v/v% alcoholic acid aqueous solution, and to regulate its pH value be 7.0.
Will be through pre-treatment (concrete pretreatment process: get a certain amount of macroporous adsorbent resin, soaked 2 hours with 2 times of left and right sides volume of ethanol, and stir frequently, make the abundant swelling of resin; Then will be fully swollen polymeric adsorbent dress post, with the flow velocity of 3～4 times of bed volume per hour, 5～8 times of resin volume of ethanol are passed through resin layer, up to behind the effluent liquid thin up constant muddy till; Resin is after Ethanol Treatment, with the flow velocity of 6～8 times of bed volume per hour with deionized water by resin layer, displace ethanol and get final product.) 12mL macroporous adsorbent resin SEPABEADS SP835 (Mitsubishi chemistry) fill in two chromatography columns in two identical chromatography columns (loadings of the diameter of chromatography column, length and macroporous adsorbent resin is all identical), they are connected in series, and are connected in series with two UV-detector baseline balances behind the chromatography column with the 5v/v% alcoholic acid aqueous solution.
(produce with connection pharmaceutcal corporation, Ltd in Yueyang with the 2.26g erythromycin, it consists of the 87.2wt% Erythromycin A, the 7.6wt% Erythromycin C, other impurity of 5.2wt%) join after being dissolved in the 30mL ethanol dilute in the certain volume water the stock liquid of the 5v/v% alcoholic acid aqueous solution of 600ml, the pH value of regulating the gained stock liquid is 6.5～7.5.Then it is joined among in above-mentioned two chromatography columns that connect with series system one (" main chromatography column ") with 1BV/h speed.With the pH value be 6.5～7.5, concentration is that the 5v/v% alcoholic acid aqueous solution is that (eluent a) for eluent, flow velocity difference wash-out " main chromatography column " and " auxilliary chromatography column " with 1BV/h, when the erythromycin concentration in " main chromatography column " effluent liquid (HPLC content) greater than 90% the time, stop wash-out, elution volume is about 4BV.Be that 6.5～7.5 aqueous ethanolic solution is eluent (eluent b) with 95v/v%, pH value then, " main chromatography column " carried out wash-out, collect elutriant with the flow velocity of 1BV/h.
Carry out chromatography column by previously described " blocked operation ", and merge the elutriant of all " main chromatography columns ".The elutriant of gained is through 50 ℃ of dry target compounds (Erythromycin A) that get.Erythromycin A purity is 91.16%, yield is not less than 87.6%.
Embodiment 2
Stock liquid and polymeric adsorbent are with embodiment 1, and institute joins and contains the 2v/v% vinyl acetic monomer in the solution, and column volume is 224ml on the stock liquid, and in addition, eluent a is the 2v/v%% vinyl acetic monomer aqueous solution, and eluent b is the saturated vinyl acetic monomer aqueous solution.The elution volume determined of Erythromycin A content in the main column effluent liquid is about 5BV after testing, and other processing step is identical with embodiment 1, and Erythromycin A purity is 95.77%, yield is not less than 83.2%.
Claims (3)
1. the method for a purification of erythromycin A is characterized in that, said method comprises the steps:
(1) with erythromycin crude product and C
1～C
3The aqueous solution of unitary fatty alcohol get stock liquid, the pH value of regulating the gained stock liquid is 6.0～9.0, break into portions is standby;
Wherein: the consisting of of erythromycin crude product: the Erythromycin A of 87.2wt%, the Erythromycin C of 7.6wt% and other impurity of 5.2wt%;
(2) macroporous adsorbent resin is filled in two diameters and the identical length chromatography column together, and it is connected in series, with C
1～C
3The solution washing of unitary fatty alcohol to being connected in series with two UV-detector baseline balances behind the chromatography column;
Wherein: said macroporous adsorbent resin is SEPABEADS SP835, and the loadings of said macroporous adsorbent resin is also identical;
To be that 6.0～9.0 stock liquid joins with in one in two chromatography columns of series system connection by a pH value of step (1) gained, be easy narration, we will be added with the pH value is that the chromatography column of 6.0～9.0 stock liquid is defined as " main chromatography column ", corresponding, another root then is called " auxilliary chromatography column ";
Wherein: by weight, the pH value is that the amount that 6.0～9.0 stock liquid is added in " main chromatography column " is 0.2～0.8 times of " main chromatography column " saturated extent of adsorption;
C with 5v/v%
1～C
3The aqueous solution of unitary fatty alcohol be eluent a, respectively " main chromatography column " and " auxilliary chromatography column " carried out wash-out with eluent a;
Adopt the concentration of the Erythromycin A in high performance liquid chromatography monitoring " main chromatography column " effluent liquid, when the concentration of Erythromycin A in " main chromatography column " effluent liquid greater than 90% the time, change eluent, promptly with the C of 95v/v%
1～C
3The aqueous solution of unitary fatty alcohol be eluent b, with eluent b " main chromatography column " carried out wash-out, collect elutriant, after drying target compound;
(3) " main chromatography column " in the step (2) regenerated and balance after connect with " auxilliary chromatography column " in the step (2) again, carry out " blocked operation " promptly then: by step (1) another part of gained pH value is that 6.0～9.0 mixed solution adds " auxilliary chromatography column " in the step (2), repeating step (2) target compound; So, " main chromatography column " and " auxilliary chromatography column " carried out " blocked operation ", and " blocked operation " is preceding each time, last round-robin " main chromatography column " must be reproduced and balance;
In with eluent a elution process, when beginning to contain Erythromycin A in " in case auxilliary chromatography column " effluent liquid, promptly finish blocked operation, and " auxilliary chromatography column " carried out wash-out with eluent b, collect elutriant, through concentrate, method such as crystallization reclaims Erythromycin A, " auxilliary chromatography column " regenerated, come into operation after the balance, begin twin columns once more and switch cyclical operation.
2. the method for claim 1 is characterized in that, wherein the pH value of stock liquid is 6.5～7.5.
3. method as claimed in claim 1 or 2 is characterized in that, wherein eluent a is the aqueous ethanolic solution of 5v/v%; Eluent b is the aqueous ethanolic solution of 95v/v%.
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| CN2008102003780A CN101367855B (en) | 2008-09-24 | 2008-09-24 | Method for purification of erythromycin A |
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|---|---|---|---|
| CN2008102003780A CN101367855B (en) | 2008-09-24 | 2008-09-24 | Method for purification of erythromycin A |
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| CN101367855B true CN101367855B (en) | 2011-05-11 |
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| CN102453063B (en) * | 2010-10-21 | 2015-04-01 | 上海医药工业研究院 | Method for separation of partricin B |
| CN102453062B (en) * | 2010-10-21 | 2015-03-04 | 上海医药工业研究院 | Partricin A separation method |
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Non-Patent Citations (1)
| Title |
|---|
| 宋应华,朱家文,陈葵,孙瑛.大孔树脂提纯红霉素的研究.《中国抗生素杂志》.2007,第32卷(第5期),284-315. * |
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