CN108250270A - A kind of method of the enrichment extraction Daptomycin from zymotic fluid - Google Patents
A kind of method of the enrichment extraction Daptomycin from zymotic fluid Download PDFInfo
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- CN108250270A CN108250270A CN201611241584.7A CN201611241584A CN108250270A CN 108250270 A CN108250270 A CN 108250270A CN 201611241584 A CN201611241584 A CN 201611241584A CN 108250270 A CN108250270 A CN 108250270A
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- daptomycin
- zymotic fluid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
The present invention relates to a kind of method of the enrichment extraction Daptomycin from zymotic fluid, step is：First by the zymotic fluid containing Daptomycin by 818 macroporous resin enrichments of HZ, then the Daptomycin that enrichment is obtained is parsed from macroreticular resin, and Daptomycin desorbed solution goes out Daptomycin semifinished product through being concentrated in vacuo with chemical solvent precipitated crystal.The low pressure resin chromatographic column that Daptomycin crude product is splined on to 300 high molecular polymerization nanoparticle fillers of RPC containing PS carries out chromatography, more than 97.5% Daptomycin mother liquor is collected in distribution, mother liquor is concentrated in vacuo again, activated carbon decolorizing, the processes such as precipitated crystal, freeze-drying obtain 98% Daptomycin sterling.The present invention has the advantages that method simplicity and technological process are suitable for producing in enormous quantities.
Description
Technical field
The invention belongs to industrial microbial technology fields, are related to the preparation method of pharmaceutical raw material, and in particular to one kind from
The preparation method of Daptomycin is isolated and purified in fermentation mycelium.
Background technology
Daptomycin is a kind of first product for being known as cyclic ester peptides antibiotics family.It is from streptomyces parvus
(Streptomyces parvus) zymotic fluid extracts the obtained cyclic lipopeptide of the structure novel of 13 amino acid composition in the middle
Antibiotic, wherein ten Amino acid profile cyclic structures, capric acid and tryptophan are esterified outside ring.It not only has novel
Chemical constitution, and its binding mode also from any to be approved antibiotic different.It can destroy bacterial cell membrane work(in many aspects
Can, gram positive bacteria is thus killed rapidly.Daptomycin is more important in addition to it can act on most of clinically relevant gram positive bacterias
Be in vitro to being in the drug resistances isolated strains such as methicillin (methic i l l in), vancomycin and Linezolid
Still has strong active.
Daptomycin is tunning, the ferment filtrate obtained by fermented and cultured, can be generated in fermentation process a large amount of
Pigment and the by-product close with Daptomycin structure are such as dehydrated Daptomycin, thus the method for extraction and purification of Daptomycin compared with
It is complicated.General Daptomycin producing strains, production capacity is unstable, and yield is relatively low, and fermentation byproduct is more, and impurity is more, causes
Extraction work is complex afterwards, substantially increases postorder purification difficulty, it is difficult to the final product of high-purity is obtained, so as to nothing
Method industrialization production.
To Daptomycin, postorder method for extraction and purification has had many reported in literature, and substantially technique is：Zymotic fluid is through super
Filter, nanofiltration, resin cation absorption pickling, resin anion (R.A.) neutralize, nanofiltration concentration, crystallization.Membrane filtration yield reaches 98%-
99%, purity can also obtain more than 98.5% after product is crystallized.But Daptomycin is an amphiprotic substance, and isoelectric point is approximately PH4-
5, under different pH conditions, its dissolubility is different, and especially there are a large amount of homologue, isomeries for Daptomycin
Body, these impurity are much like with Daptomycin in nature, therefore, industrially to be separated pure by so simple technique
It is highly difficult to change even crystallization Daptomycin.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of enrichment extraction Daptomycin from zymotic fluid
Method.
Technical scheme is as follows：The present invention provides a kind of method of the enrichment extraction Daptomycin from zymotic fluid,
Include the following steps：
A. first Daptomycin in zymotic fluid is enriched with, then obtained Daptomycin coarse extraction will be enriched with, wherein coarse extraction is adopted
The neutrality of zymotic fluid PH to 7.5 is adjusted with 0.1% hydrochloric acid, is used in combination zymotic fluid by HZ-818 macroporous resin adsorptions after press filtration
The impurity such as polysaccharide protein of the ethanol elution absorption of 50%-75%, then with 75%-90% ethanol solution gradient elutions, must reach
Tobramycin zymotic fluid.
B. obtained Daptomycin zymotic fluid rotary evaporator liquid is removed in zymotic fluid after ethanol solution with 0.1% salt
Acid solution adjusts PH to 7.5；
C. the Daptomycin obtained by precipitation vessel in settling step b, obtains crystal, and stir the crystal slurry,
And pH value is continuously adjusted to required degree, until precipitation completely, obtains crystal precipitation；
D. Daptomycin crude product is obtained by freeze-drying Daptomycin preparation solution.
E., Daptomycin crude product is splined on to the low pressure resin layer of the nanoparticle filler of high molecular polymerization containing PS-RPC-300
It analyses column and carries out chromatography, distribution is collected more than 98% Daptomycin mother liquor, mother liquor is concentrated in vacuo again, activated carbon takes off
The processes such as color, precipitated crystal, freeze-drying obtain 98% Daptomycin sterling.
It is currently preferred, according in step c, miscible with water have by being added in containing net antibiotic activity eluent
Solvent crystallizes, and Daptomycin content is 60%~75% in solution before crystallization.According in step e, by containing net antibiosis
Organic solvent miscible with water is added in plain active eluant to crystallize, before crystallization in solution Daptomycin content for 97%~
98%.
Currently preferred, the solvent is methanol, ethyl alcohol, isopropanol or acetone.
Invention is preferred, and the solvent addition is：Methanol is 2~6 times of crystal solution volumes；Ethyl alcohol is 1.5~5.5 times
Crystal solution volume；Isopropanol is 1.2~5 times of crystal solution volumes；Acetone is 1.0~4.5 times of crystal solution volumes.
Currently preferred, according in step c, crystallization process initial temperature is 25~60C °；Crystallization process outlet temperature
It is 0~30C °；Crystallization process cooling rate is 0.5~5C °/minute.
Beneficial effects of the present invention are as follows：
Using the above scheme, for the present invention by press filtration membrane filtration, obtained clear filtrate passes through macroporous absorption chromatography resin
Column, Daptomycin are attracted on macroporous absorption chromatography resin column, and the resin of adsorption saturation is parsed through ethyl alcohol, and precipitation process is easily-controllable
System, mixed liquor easily filter, and obtained Daptomycin is of light color, and crude product is through PS-RPC-300 high molecular polymerization nanoparticle fillers
The chromatography of low pressure resin chromatographic column, fraction collection are concentrated in vacuo, activated carbon decolorizing, precipitated crystal, the processing such as freeze-drying
Afterwards, powder is loose, soluble；The present invention has the advantages that intermediate processing simplicity and technological process are suitable for producing in enormous quantities.
Description of the drawings
Fig. 1 is a kind of flow chart of the method for enrichment extraction Daptomycin from zymotic fluid of the present invention:
Fig. 2 is the HPLC liquid chromatograms of Daptomycin after purification.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Referring to Fig. 1, the present invention provides a kind of method of the enrichment extraction Daptomycin from zymotic fluid, including following step
Suddenly：
A. the HZ-818 of Daptomycin in zymotic fluid is first adsorbed into resin concentration, then obtained Daptomycin crude product will be enriched with
By extraction, ethyl alcohol or acetone soln in zymotic fluid are removed including Daptomycin zymotic fluid rotary evaporator will be enriched with,
Organic solvent miscible with water is added dropwise to crystallize in Daptomycin mother liquor after concentration, precipitation is mould up to holding in the palm in precipitation vessel
Element obtains Daptomycin crystal, and stirs the crystal slurry, has controlled temperature, until precipitation completely, obtains crystal precipitation；
Daptomycin crude product is obtained by being freeze-dried Daptomycin crystal.B. Daptomycin crude product is splined on containing PS-RPC-300 high
The low pressure resin chromatographic column of molecule aggregation nanoparticle filler carries out chromatography, and it is female that more than 97.5% Daptomycin is collected in distribution
Liquid is concentrated in vacuo mother liquor, activated carbon decolorizing, precipitated crystal again, and the processes such as freeze-drying i.e. available 98% reaches support
Mycin sterling.
Embodiment one：
The present invention includes the following steps：
A. 0.1% hydrochloric acid of zymotic fluid 100L that spare potency containing Daptomycin is 521ug/ml is adjusted into zymotic fluid PH=
7.5 is neutral, and mycelium is removed with the cloth bag filter press press filtration of 0.02u membrane apertures, collects filtrate, and the 5.2Kg mycelium of collection are used
10L absolute ethyl alcohols dissolve and stir 1h, remove mycelium with the cloth bag filter press press filtration of 0.02u membrane apertures again, will contain up to support
The ethanol solution of mycin is incorporated in the filtrate collected after press filtration early period, and filtrate volume is 107.5L. after merging
B. the zymotic fluid of the 107.5L collected after press filtration is fitted into the head tank of 200L volumes, usedFor 150*
The stainless steel chromatographic column of 1500mm makees HZ-818 large pore resin absorption columns, and the control of ferment filtrate adsorption flow rate is in 25L/h, control 4h
Absorption terminates.
C. continue gradient elution with 50% -75% ethanol solutionStainless steel chromatographic column for 150*1500mm makees HZ-
818 large pore resin absorption columns, elution flow rate control is in 25L/h, until terminating to elute during lower prop solution colorless clear, Ran Houyong
The Daptomycin of absorption is parsed lower prop by 75L80% acetone solns, is added up to and is collected filtrate 72L.
D. filtrate of the 72L collections containing Daptomycin under 40C ° is concentrated in vacuo with rotary evaporator, removes ethyl alcohol
With partially aqueous solution to 2.5L, in the 2.5L mother solution displacements containing Daptomycin to 20L precipitation vessels, will be added dropwise miscible with water has
Solvent crystallizes, and Daptomycin is precipitated in precipitation vessel, obtains Daptomycin crystal, and the solvent addition is：First
Alcohol is 4 times of crystal solution volumes；Ethyl alcohol is 3.5 times of crystal solution volumes；Isopropanol is 2.3 times of crystal solution volumes；Acetone is 3.3 times of knots
Brilliant liquid product stirs the crystal slurry, and crystallization initial temperature is set as 45C ° in the process, and terminal point control temperature is 10C °, knot
Brilliant process cooling rate is 2C °/minute.Until precipitation completely after being added dropwise, the precipitated crystal time in 12h, obtains crystal and sinks
Starch；By filtering, 81.6g Daptomycin crude products are obtained after freeze-drying, are about 65% through analyzing Daptomycin purity.
E. by purity 65%, 81g Daptomycins crude product is fully dissolved with 400 milliliter of 80% acetone, is splined onFor
The low pressure resin chromatographic column of 150*1500mm high molecular polymerizations containing PS-RPC-300 nanoparticle is chromatographed, first with 50 liters
20% acetone starts to elute as eluant, eluent, and flow control is in 5ml/min, collection eluent, then is made with 50 liter 30% of acetone
Eluant, eluent starts to elute, and flow control is collected using distribution, purity up to 97.5% eluent is collected, is finally used in 5ml/min
50 liter 40% of acetone starts to elute as eluant, eluent, and for flow control in 5ml/min, more than 97.5% Daptomycin mother is collected in distribution
Liquid merges more than 97.5% Daptomycin mother liquor and carries out being concentrated under reduced pressure into 2.5L, with 20g powder activity carbon decoloring 1h, be added dropwise with
The organic solvent that water dissolves each other crystallizes, and Daptomycin is precipitated in precipitation vessel, obtains Daptomycin crystal, and the solvent adds
Entering amount is：Methanol is 4 times of crystal solution volumes；Ethyl alcohol is 3.5 times of crystal solution volumes；Isopropanol is 2.3 times of crystal solution volumes；Acetone
For 3.3 times of crystal solution volumes, the crystal slurry is stirred in the process, crystallization initial temperature is 45C °, and terminal point control temperature is 10C °,
Crystallization process cooling rate is 2C °/minute.Until precipitation completely after being added dropwise, the precipitated crystal time in 12h, obtains crystal
Sediment；By filtering, 39.94g Daptomycin sterlings are obtained after freeze-drying, are about through analyzing Daptomycin purity
98.2%.
In conclusion using the above scheme, it is of the invention by Daptomycin in HZ-818 macroporous resin adsorption zymotic fluids, then
Obtained Daptomycin will be enriched with by parsing and extraction acquisition Daptomycin crude product, Daptomycin crude product is splined on containing PS-
The low pressure resin chromatographic column of RPC-300 high molecular polymerization nanoparticle fillers carries out chromatography, and distribution collects more than 97.5%
Daptomycin mother liquor is concentrated in vacuo mother liquor, activated carbon decolorizing, precipitated crystal again, and the processes such as freeze-drying can obtain
To 98% Daptomycin sterling；The present invention, which has to the Daptomycin in zymotic fluid to have, to collect efficient, and loss is few, and purifying, which reaches, holds in the palm
The advantages of mycin method simplicity and technological process are suitable for producing in enormous quantities.
The foregoing is merely a prefered embodiment of the invention, is not intended to restrict the invention, it is all the present invention spirit and
All any modification, equivalent and improvement made within principle etc., should all be included in the protection scope of the present invention.
Claims (6)
- A kind of 1. method of the enrichment extraction Daptomycin from zymotic fluid, which is characterized in that include the following steps：A. first Daptomycin in zymotic fluid is enriched with, then obtained Daptomycin coarse extraction will be enriched with, wherein coarse extraction uses 0.1% hydrochloric acid adjusts zymotic fluid PH=7.5, removes mycelium, then adsorbed with HZ-816 macroporous resin columns with filter press press filtration, inhales It is attached to be parsed again, obtain Daptomycin enrichment zymotic fluid.B. the Daptomycin obtained by precipitation vessel in settling step a, obtains crystal, and stir the crystal slurry, by PH Value is continuously adjusted to required degree, until precipitation completely, obtains crystal precipitation；C. Daptomycin is obtained by freeze-drying Daptomycin preparation solution.D., Daptomycin crude product is splined on to the low pressure resin chromatographic column of the nanoparticle filler of high molecular polymerization containing PS-RPC-300 Chromatography is carried out, distribution is collected more than 97.5% Daptomycin mother liquor, mother liquor is concentrated in vacuo again, activated carbon takes off The processes such as color, precipitated crystal, freeze-drying obtain 98% Daptomycin sterling.
- 2. a kind of method of enrichment extraction Daptomycin from zymotic fluid according to claim 1, which is characterized in that according to In step b, crystallized by adding in organic solvent miscible with water in containing net antibiotic activity eluent, before crystallization in solution Daptomycin content is 50%~65%.
- 3. a kind of method of enrichment extraction Daptomycin from zymotic fluid according to claim 1, which is characterized in that according to In step d, crystallized by adding in organic solvent miscible with water in containing net antibiotic activity eluent, before crystallization in solution Daptomycin content is 97%~98%.
- 4. the method for a kind of enrichment extraction Daptomycin from zymotic fluid according to claim 2, which is characterized in that described Solvent is methanol, ethyl alcohol, isopropanol or acetone.
- 5. the method for a kind of enrichment extraction Daptomycin from zymotic fluid according to claim 3, which is characterized in that described Solvent addition is：Methanol is 2~6 times of crystal solution volumes；Ethyl alcohol is 1.5~5.5 times of crystal solution volumes；Isopropanol is 1.2 ~5 times of crystal solution volumes；Acetone is 1.0~4.5 times of crystal solution volumes.
- 6. a kind of method of enrichment extraction Daptomycin from zymotic fluid according to claim 1, which is characterized in that according to In step b, d, crystallization process initial temperature is 45 DEG C；Crystallization process outlet temperature is 10 DEG C；Crystallization process cooling rate is 0.5 DEG C~5 DEG C/min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117311A (en) * | 2019-04-30 | 2019-08-13 | 湖南师范大学 | A method of isolating and purifying bacillomycin from bacillus amyloliquefaciens |
| CN110117310A (en) * | 2019-04-17 | 2019-08-13 | 华北制药华胜有限公司 | A kind of purification process of Daptomycin |
| CN114344447A (en) * | 2021-12-16 | 2022-04-15 | 华北制药股份有限公司 | Daptomycin for injection and preparation method thereof |
| CN115417822A (en) * | 2022-08-17 | 2022-12-02 | 山东福瑞达生物科技有限公司 | Extraction and purification process of tetrahydropyrimidine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117310A (en) * | 2019-04-17 | 2019-08-13 | 华北制药华胜有限公司 | A kind of purification process of Daptomycin |
| CN110117311A (en) * | 2019-04-30 | 2019-08-13 | 湖南师范大学 | A method of isolating and purifying bacillomycin from bacillus amyloliquefaciens |
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| CN114344447A (en) * | 2021-12-16 | 2022-04-15 | 华北制药股份有限公司 | Daptomycin for injection and preparation method thereof |
| CN114344447B (en) * | 2021-12-16 | 2023-08-25 | 华北制药股份有限公司 | Daptomycin for injection and preparation method thereof |
| CN115417822A (en) * | 2022-08-17 | 2022-12-02 | 山东福瑞达生物科技有限公司 | Extraction and purification process of tetrahydropyrimidine |
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