CN102276696A - Method for purifying daptomuycin - Google Patents
Method for purifying daptomuycin Download PDFInfo
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- CN102276696A CN102276696A CN2010101962477A CN201010196247A CN102276696A CN 102276696 A CN102276696 A CN 102276696A CN 2010101962477 A CN2010101962477 A CN 2010101962477A CN 201010196247 A CN201010196247 A CN 201010196247A CN 102276696 A CN102276696 A CN 102276696A
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Abstract
The invention discloses a method for purifying daptomuycin. According to the method, half-purified daptomuycin is loaded on a gel type weak anion resin for combination at first and is eluted the next. The purification method provided in the invention enables obtained daptomuycin to have purity higher than 98%; the raw materials used in the method are easily available and cheap; therefore, it is easy to popularize and apply the method.
Description
Technical field
The present invention relates to medicine purifying field, be specifically related to a kind of purification process of daptomycin.
Background technology
The trend that is caused infection by the Gram-positive resistant organism is in continuous rising.Though glycopeptide antibiotics such as vancomycin are to these pathogenic bacteria of drug-resistant, has certain result of treatment as methicillin-resistant staphylococcus aureus, epidermis grape ball and faecalis persister etc., but in recent years, along with the appearance of vancomycin resistance gram resistant organism clinically, make this trend more increase, therefore press for searching one class antibiosis and usually resist resistant organism.
Daptomycin is a kind of microbiotic that is produced by Streptomyces roseosporus, as shown in Figure 1, form by 13 amino acid, wherein ten amino acid are formed an amino acid peptide ring, other has three amino acid to be arranged in wire, L-tryptophane terminal in these three amino acid is followed one ten carbon capric acid, is a kind of microbiotic of calcium ion dependent form.Under the condition that calcium ion exists, it will combine with daptomycin conjugated protein (DBPs) on the cytolemma with the form of non covalent bond.Daptomycin has the good sterilization effect for gram-positive microorganism, can be developed into preparation and is used to the infection that includes but not limited to that methicillin-resistant staphylococcus glucose fungus and vancomycin-resistant enterococcus cause.
Divide an application (application number 200810087401.X) and U.S. Pat 6696412B1 etc. of Chinese invention patent application (application number 01805212.6) and its disclose the purification process of daptomycin.Utilize resin anion(R.A) FP-DA13 enrichment daptomycin from fermented liquid, then utilize reversed-phase resin HP-20ss to be further purified daptomycin, obtain semipurified daptomycin.Then semipurified daptomycin is crossed Poros P150 or Poros D50 (Applied Biosystem) purifying, obtained purity and be 98% daptomycin.This method provides the method for the feasible separation daptomycin of a kind of commercialization.But, Poros P150 and two kinds of resins of Poros D50 of having used in the method, produce by Applied Biosystem company, its price is very expensive, though separating effect is better, but, in the separation and purification process, need very big pressure just can reach certain flow velocity because its particle is very thin.
Summary of the invention
The purification process that the objective of the invention is to centering state application for a patent for invention (application number 01805212.6) and its disclosed daptomycins such as (application number 200810087401.X) and U.S. Pat 6696412B1 of dividing an application is done further to improve, to reduce cost, be beneficial to industrialization promotion and use.
For this reason, the invention provides a kind of purification process of daptomycin, comprise the step that semipurified daptomycin is further purified, concrete, described purifying is meant described semipurified daptomycin loaded and carries out wash-out after being attached to the gel-type weakly anionic resin.
The purification process of daptomycin of the present invention, described gel-type weakly anionic resin is a dextrane gel type weakly anionic resin, described dextrane gel type weakly anionic resin is dextrane gel A25, described dextrane gel A25 adopts the acetate buffer solution or the Tris damping fluid balance of the urea that contains 0-4mol/L before use, and the pH of described damping fluid is 6～8.Described semipurified daptomycin carries out wash-out after loading and being attached to dextrane gel A25, after using the acetate buffer solution or the washing of Tris damping fluid of volume as the urea that contains 0-4mol/L of 3 times of column volumes, the employing volume is that the acetate buffer solution that contains 0.1-0.5mol/L sodium-chlor, 0-4mol/L urea or the Tris damping fluid of 10 times of column volumes carries out wash-out, and the pH of described damping fluid is 6-8.
The purification process of daptomycin of the present invention, described gel-type weakly anionic resin is a sepharose type weakly anionic resin, described sepharose type weakly anionic resin is DEAE fast flow, described DEAE fastflow adopts the acetate buffer solution or the Tris damping fluid balance of the urea that contains 0-6mol/L before use, and the pH of described damping fluid is 6～8.Described semipurified daptomycin loads to be attached on the described DEAE fast flow and carries out separation and purification, after using the acetate buffer solution that contains 0-6mol/L urea or the washing of Tris damping fluid of volume as 3 times of column volumes, it is that the acetate buffer solution that contains 0.015-0.3mol/L sodium-chlor, 0-6mol/L urea or the Tris damping fluid of 5～10 times of column volumes carries out wash-out that employing contains volume, and the pH of described damping fluid is 6-8.
The purification process of daptomycin of the present invention, described semipurified daptomycin are through step: A), adopt negatively charged ion to count fat thick solution of enrichment daptomycin from the daptomycin fermented liquid; B), adopt the thick solution of the described daptomycin of reverse resin purification to make.
Purification process of the present invention, the gel type resin of use, raw material be easy to get, relatively inexpensive, be easy to industrialization promotion and use.In damping fluid, add urea, destroy non covalent bond, help wash-out daptomycin from the resin.Purification process of the present invention, the purity of the daptomycin that obtains is higher than 98%.
Description of drawings
Fig. 1 is the daptomycin structure iron.
Fig. 2 is that the HPLC of daptomycin behind the purifying detects collection of illustrative plates.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples only are used to the present invention to be described but not to be used to limits the scope of the invention.
In the present invention, the HPLC condition is as follows:
Moving phase: A phase: second eyeball: damping fluid (10: 90)
B phase: second eyeball: damping fluid (45: 55)
Damping fluid: 0.5% (NH
4) H
2PO
4
Gradient (18min)
Chromatographic column: Hypersil C18,4.6*250mm, 5 μ m
Column temperature: 35 ℃
Detector: DAD (HP1100) detector
Detect wavelength: 223nm
Flow velocity: 1.5ml/min
Sample size: 5 μ l
Type of elution: as shown in table 1.
Table 1, HPLC elution protocol
| Elution time (min) | 0 | 15 | 18 |
| A(％) | 25 | 0 | 0 |
| B(％) | 75 | 100 | 100 |
Embodiment 1 semipurified daptomycin
With reference to Chinese invention patent application (application number 01805212.6) and its divide an application (application number 200810087401.X) and U.S. Pat 6696412B1 disclosed method, prepare semipurified daptomycin, specific as follows:
At controlled condition bottom fermentation Streptomyces roseosporus, obtain the daptomycin fermented liquid.Carry out described fermented liquid centrifugal or filtration, obtain clear soln.With the FP-DA13 of anionite-exchange resin Mitsubishi on the clarifying fermented liquid, under the pH6.5 condition, wash then with 0.03mol/L NaCl, and at pH6.5 condition 0.3mol/L NaCl wash-out.Elutriant is loaded on the reversed-phase resin HP-20ss, cleans the HP-20ss post, and,, obtain liquid phase purity and be about 90% semipurified daptomycin solution through the HPLC detection with the Virahol wash-out of 55-60% with 45% Virahol.
The daptomycin of embodiment 2 purifying
Obtaining liquid phase purity by embodiment 1 method is 90% semipurified daptomycin solution, and it is loaded on the dextrane gel A25, and A25 adopts pH7.0 acetate buffer solution balance in advance.After treating that daptomycin is attached to resin, use pH 7.0 acetate buffer solutions of 3 times of column volumes to clean, use the pH that contains 0.5mol/L NaCl 7.0 acetate buffer solutions of 10 times of column volumes to carry out wash-out then, obtain the daptomycin of purifying.Adopt HPLC to detect the purity of daptomycin, the result is 98.6%, as shown in Figure 2.
The daptomycin of embodiment 3 purifying
The liquid phase purity that obtains by embodiment 1 method is 91% semipurified daptomycin solution, and it is loaded on the dextrane gel A25, and A25 has used the pH 6.0 acetate buffer solution balances that contain 2mol/L urea in advance.After treating that daptomycin is attached on the resin, use 3 times of column volumes to contain the pH 6.0 acetate buffer solutions washing of 2mol/L urea, use pH 6.0 acetate buffer solutions that contain 0.2mol/L NaCl, 2mol/L urea of 10 times of column volumes to carry out wash-out then, detect through HPLC, obtain purity and be 98.5% daptomycin.
The daptomycin of embodiment 4 purifying
Obtaining liquid phase purity by embodiment 1 method is 88% daptomycin solution, and it is loaded on the dextrane gel A25, and A25 has used the pH 8.0Tris damping fluid balance that contains 4mol/L urea in advance.After treating that daptomycin is attached on the resin, use 3 times of column volumes to contain the pH 8.0Tris damping fluid washing of 4mol/L urea, use the pH 8.0Tris damping fluid that contains 0.1mol/L NaCl, 4mol/L urea of 10 times of column volumes to carry out wash-out then, detect through HPLC, obtain purity and be 98.2% daptomycin.
The daptomycin of embodiment 5 purifying
Obtaining liquid phase purity by embodiment 1 method is 89% daptomycin solution.This solution of 50ml is loaded on the 20ml sepharose DEAE Fast Flow, and DEAE Fast Flow adopts the acetate buffer solution balance of the pH 6.5 that contains 2mol/L urea in advance.After treating that daptomycin all is loaded on the resin, use the pH that the contains 2mol/L urea 6.5 acetate buffer solutions washing of 3 times of column volumes, pH 6.5 acetate buffer solutions that adopt 100ml to contain 0.1mol/LNaCl, 2mol/L urea afterwards carry out wash-out, detect through HPLC, obtain purity and be 98.9% daptomycin.
The daptomycin of embodiment 6 purifying
Obtaining liquid phase purity according to embodiment 1 described method is 92% daptomycin solution.This solution of 1000ml is loaded on the 100ml sepharose DEAE Fast Flow, and DEAE Fast Flow adopts the Tris damping fluid balance of the pH 7.5 that contains 1mol/L urea in advance.After treating that daptomycin all is loaded on the resin, use the pH 7.5Tris damping fluid washing that contains 1mol/L urea of 3 times of column volumes, the pH 7.5Tris damping fluid that adopts 1000ml to contain 0.15mol/L NaCl, 1mol/L urea afterwards carries out wash-out, detect through HPLC, obtain purity and be 98.7% daptomycin.
The daptomycin of embodiment 7 purifying
Obtaining liquid phase purity according to embodiment 1 described method is 92% daptomycin solution.This solution of 1000ml is loaded on the 100ml sepharose DEAE Fast Flow, and DEAE Fast Flow adopts the Tris damping fluid balance of the pH 8.0 that contains 4mol/L urea in advance.After treating that daptomycin all is loaded on the resin, use the pH 8.0Tris damping fluid washing that contains 4mol/L urea of 3 times of column volumes, the pH 8.0Tris damping fluid that adopts 1000ml to contain 0.05mol/L NaCl, 4mol/L urea afterwards carries out wash-out, detect through HPLC, obtain purity and be 98.8% daptomycin.
The daptomycin of embodiment 8 purifying
Obtaining liquid phase purity according to embodiment 1 described method is 92% daptomycin solution.This solution of 500ml is loaded on the 50ml sepharose DEAE Fast Flow, and DEAE Fast Flow adopts the acetate buffer solution balance of the pH 6.0 that contains 6mol/L urea in advance.After treating that daptomycin all is loaded on the resin, use the pH that the contains 6mol/L urea 6.0 acetate buffer solutions washing of 3 times of column volumes, pH 6.0 acetate buffer solutions that adopt 500ml to contain 0.015mol/L NaCl, 6mol/L urea afterwards carry out wash-out, detect through HPLC, obtain purity and be 98.7% daptomycin.
The daptomycin of embodiment 9 purifying
Obtaining liquid phase purity according to embodiment 1 described method is 91% daptomycin solution.This solution of 1000ml is loaded on the 100ml sepharose DEAE Fast Flow, and DEAE Fast Flow adopts the acetate buffer solution balance of pH6.5 in advance.After treating that daptomycin all is loaded on the resin, use the pH6.5 acetate buffer solution washing of 3 times of column volumes, pH 6.5 acetate buffer solutions that adopt 1000ml to contain 0.3mol/L NaCl afterwards carry out wash-out, detect through HPLC, obtain purity and be 98.5% daptomycin.
Purification process of the present invention, compared with prior art, the purity that obtains daptomycin is higher than 98%, in purge process, the gel type resin that uses, Poros P150 or the Poros D50 price used compared to existing technology are much lower, and the market price has only 1/4th of Poros P150 and Poros D50, effectively reduces production costs.Meanwhile, the gel type resin source that the present invention uses is abundant, can provide business-like product by how tame supplier.And Poros P150 and Poros D50 are only provided by Appied Biosystem company, and it is single to originate, and has limited applying of method.In addition, though commercially available Poros P150 and Poros D50 resin can bear very big pressure reduction, compare the gel type resin that uses among the present invention, its flow velocity is much lower under identical pressure.Therefore, use gel type resin in the present invention, be more conducive to purification process industrialization promotion of the present invention and use.In a word, the invention provides that a kind of raw material is easy to get, relatively inexpensive, the purification process that is easy to the daptomycin that industrialization promotion uses.
Claims (10)
1. the purification process of a daptomycin comprises the step that semipurified daptomycin is further purified, and it is characterized in that, described purifying is meant described semipurified daptomycin loaded and carries out wash-out after being attached to the gel-type weakly anionic resin.
2. purification process according to claim 1 is characterized in that, described gel-type weakly anionic resin is a dextrane gel type weakly anionic resin.
3. purification process according to claim 2 is characterized in that, described dextrane gel type weakly anionic resin is dextrane gel A25.
4. purification process according to claim 3 is characterized in that, described dextrane gel A25 adopts the acetate buffer solution or the Tris damping fluid balance of the urea that contains 0-4mol/L before use, and the pH of described damping fluid is 6～8.
5. purification process according to claim 3, it is characterized in that, after described wash-out is meant and uses the acetate buffer solution or the washing of Tris damping fluid of volume as the urea that contains 0-4mol/L of 3 times of column volumes, the employing volume is that the acetate buffer solution that contains 0.1-0.5mol/L sodium-chlor, 0-4mol/L urea or the Tris damping fluid of 10 times of column volumes carries out wash-out, and the pH of described damping fluid is 6-8.
6. purification process according to claim 1 is characterized in that, described gel-type weakly anionic resin is a sepharose type weakly anionic resin.
7. purification process according to claim 6 is characterized in that, described sepharose type weakly anionic resin is DEAE fast flow.
8. purification process according to claim 7 is characterized in that, described DEAE fast flow adopts the acetate buffer solution or the Tris damping fluid balance of the urea that contains 0-6mol/L before use, and the pH of described damping fluid is 6～8.
9. purification process according to claim 7, it is characterized in that, after described wash-out is meant and uses the acetate buffer solution that contains 0-6mol/L urea or the washing of Tris damping fluid of volume as 3 times of column volumes, it is that the acetate buffer solution that contains 0.015-0.3mol/L sodium-chlor, 0-6mol/L urea or the Tris damping fluid of 5～10 times of column volumes carries out wash-out that employing contains volume, and the pH of described damping fluid is 6-8.
10. purification process according to claim 1 is characterized in that, described semipurified daptomycin makes through following steps:
A), adopt negatively charged ion to count fat thick solution of enrichment daptomycin from the daptomycin fermented liquid;
B), adopt the thick solution of the described daptomycin of reverse resin purification.
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| CN201010196247.7A CN102276696B (en) | 2010-06-09 | 2010-06-09 | Method for purifying daptomuycin |
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| CN102718839A (en) * | 2012-07-05 | 2012-10-10 | 鲁南新时代生物技术有限公司 | Method for separating and purifying daptomycin |
| CN105001305A (en) * | 2015-04-29 | 2015-10-28 | 利穗科技(苏州)有限公司 | Method for extracting high-purity daptomycin by utilizing chromatographic technique |
| WO2019233417A1 (en) * | 2018-06-04 | 2019-12-12 | 浙江医药股份有限公司新昌制药厂 | Spray-dried powder containing daptomycin and industrial preparation method therefor |
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| CN105001305A (en) * | 2015-04-29 | 2015-10-28 | 利穗科技(苏州)有限公司 | Method for extracting high-purity daptomycin by utilizing chromatographic technique |
| WO2019233417A1 (en) * | 2018-06-04 | 2019-12-12 | 浙江医药股份有限公司新昌制药厂 | Spray-dried powder containing daptomycin and industrial preparation method therefor |
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