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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- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 title claims abstract description 88
- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 86
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000012530 fluid Substances 0.000 title claims abstract description 33
- 238000000605 extraction Methods 0.000 title claims abstract description 22
- 239000013078 crystal Substances 0.000 claims abstract description 40
- 230000008569 process Effects 0.000 claims abstract description 19
- 239000011347 resin Substances 0.000 claims abstract description 18
- 229920005989 resin Polymers 0.000 claims abstract description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000012043 crude product Substances 0.000 claims abstract description 12
- 239000012452 mother liquor Substances 0.000 claims abstract description 11
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 238000009826 distribution Methods 0.000 claims abstract description 7
- 239000002105 nanoparticle Substances 0.000 claims abstract description 7
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 6
- 239000000945 filler Substances 0.000 claims abstract description 6
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 26
- 235000019441 ethanol Nutrition 0.000 claims description 16
- 238000001556 precipitation Methods 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000002425 crystallisation Methods 0.000 claims description 15
- 230000008025 crystallization Effects 0.000 claims description 15
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 230000003115 biocidal effect Effects 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 5
- 239000002002 slurry Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 239000011265 semifinished product Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 20
- 238000010521 absorption reaction Methods 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 238000010828 elution Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 241000192125 Firmicutes Species 0.000 description 2
- 241000933218 Streptomyces parvus Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000012539 chromatography resin Substances 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 238000001728 nano-filtration Methods 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- -1 cyclic ester Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229960003907 linezolid Drugs 0.000 description 1
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000005554 pickling Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229960000707 tobramycin Drugs 0.000 description 1
- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
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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| CN201611241584.7A CN108250270A (en) | 2016-12-29 | 2016-12-29 | A kind of method of the enrichment extraction Daptomycin from zymotic fluid |
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| CN201611241584.7A CN108250270A (en) | 2016-12-29 | 2016-12-29 | A kind of method of the enrichment extraction Daptomycin from zymotic fluid |
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| 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 |
| 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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| 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 |
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| 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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