CN113004373B - Daptomycin preparation method Process for purifying a substance - Google Patents
Daptomycin preparation method Process for purifying a substance Download PDFInfo
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- CN113004373B CN113004373B CN201911317729.0A CN201911317729A CN113004373B CN 113004373 B CN113004373 B CN 113004373B CN 201911317729 A CN201911317729 A CN 201911317729A CN 113004373 B CN113004373 B CN 113004373B
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- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 110
- 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 110
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 109
- 238000000034 method Methods 0.000 title claims abstract description 43
- 239000000126 substance Substances 0.000 title description 3
- 238000002360 preparation method Methods 0.000 title description 2
- 239000011347 resin Substances 0.000 claims abstract description 102
- 229920005989 resin Polymers 0.000 claims abstract description 102
- 238000001728 nano-filtration Methods 0.000 claims abstract description 82
- 239000000919 ceramic Substances 0.000 claims abstract description 35
- 239000012528 membrane Substances 0.000 claims abstract description 33
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 31
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 25
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- 238000004108 freeze drying Methods 0.000 claims abstract description 21
- 239000002158 endotoxin Substances 0.000 claims abstract description 17
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 14
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 14
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 93
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 62
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 48
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 48
- 239000001632 sodium acetate Substances 0.000 claims description 48
- 235000017281 sodium acetate Nutrition 0.000 claims description 48
- 239000007788 liquid Substances 0.000 claims description 45
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 38
- 238000011068 loading method Methods 0.000 claims description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 238000005406 washing Methods 0.000 claims description 26
- 239000011780 sodium chloride Substances 0.000 claims description 19
- 239000008215 water for injection Substances 0.000 claims description 16
- 238000007865 diluting Methods 0.000 claims description 13
- 239000004695 Polyether sulfone Substances 0.000 claims description 8
- 238000007710 freezing Methods 0.000 claims description 8
- 230000008014 freezing Effects 0.000 claims description 8
- 229920006393 polyether sulfone Polymers 0.000 claims description 8
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims 1
- 238000011033 desalting Methods 0.000 abstract description 10
- 238000000746 purification Methods 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 34
- 239000012466 permeate Substances 0.000 description 25
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 238000010521 absorption reaction Methods 0.000 description 18
- 238000001514 detection method Methods 0.000 description 17
- 238000005374 membrane filtration Methods 0.000 description 14
- 239000008213 purified water Substances 0.000 description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- 238000012544 monitoring process Methods 0.000 description 12
- 230000001105 regulatory effect Effects 0.000 description 11
- 239000003480 eluent Substances 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 241000239218 Limulus Species 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 7
- 229960000707 tobramycin Drugs 0.000 description 7
- 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 7
- 238000011010 flushing procedure Methods 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- 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 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229960003085 meticillin Drugs 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 108010059993 Vancomycin Proteins 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229960003907 linezolid Drugs 0.000 description 2
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229960003165 vancomycin Drugs 0.000 description 2
- 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 2
- 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 2
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000958215 Streptomyces filamentosus Species 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 229940021392 cubicin Drugs 0.000 description 1
- 229940032301 daptomycin injection Drugs 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 238000011013 endotoxin removal Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 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 invention belongs to the technical field of biological medicines, and particularly discloses a method for purifying daptomycin. The method comprises the following steps: 1) Filtering the daptomycin fermentation liquor by a ceramic membrane; 2) Carrying out hydrophobic resin chromatography; 3) Ion exchange resin chromatography; 4) Desalting by hydrophobic resin chromatography and removing endotoxin; 5) Nanofiltration and concentration; 6) And (5) freeze drying. The purification method of daptomycin provided by the invention has the advantages of simple operation, short steps, short production period, high product yield and high purity of daptomycin, and is beneficial to large-scale production.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for purifying daptomycin.
Background
Daptomycin is a cycloaliphatic peptide substance extracted from fermentation broth of Streptomyces roseosporus, and was originally developed in the 80 s by Lily corporation in the United states, 11 months 1997, which assigned the global exclusive development, production and sales rights of daptomycin to Cubist pharmaceutical company, 9/12/2003, and the first approval of Cubicin (Daptomycin injection) by the FDA. It is the first cyclic lipopeptid antibiotic drug to be marketed for the treatment of concurrent skin and skin structure infections caused by some gram-positive sensitive strains. Daptomycin was marketed in australia, germany, the netherlands, spanish, united kingdom in 2006 and in swiss in 2007. Then registered in 10 or more countries of canada, korea, european union, etc. U.S. FDA approval again at 3 2006; new indications for daptomycin-for the treatment of methicillin-sensitive staphylococcus aureus (MSSA) or methicillin-resistant staphylococcus aureus (MRSA) bacteremia and right endocarditis caused thereby.
Linezolid (linezolid), developed by Pfizer corporation in 2000, was only one of two new structural antibiotics developed in the last forty years, and reports of cross-resistant bacteria have appeared in less than one year. Daptomycin, in turn, has several unique properties that can confer clinical advantages. Firstly, the action mechanism of daptomycin is different from that of various antibacterial drugs on the market, which means that daptomycin will not be affected by cross drug resistance caused by other antibiotics, daptomycin is used as the first cyclic lipopeptide antibiotic applied to clinic, cross drug resistant bacteria have not been reported so far, and the occurrence probability of clinical drug resistant strains is extremely low (< 0.2%). Second, daptomycin has been shown to rapidly inhibit most clinical gram-positive cocci in vitro, including methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, and vancomycin-resistant staphylococcus aureus. Third, daptomycin only needs to be administered 1 time a day, compared to at least 2 times a day of traditional antibiotics, the dosage regimen is significantly simplified and medical cost expenditures can be effectively reduced. Based on the factors, the medicine has wide market prospect in marketing and sales.
At present, some purification processes of daptomycin are reported at home and abroad. Chinese patent CN1404487 filed by the united states casster pharmaceutical company discloses a method for purifying daptomycin by using anion resin, which alternately uses anion resin FP-DA13, macroporous resin HP-20SS, anion resin PorosD or Poros 150 to separate and purify daptomycin, thus a daptomycin product with purity of 98% can be obtained, but the process is complicated, the cost is high, and industrial scale production is difficult to realize.
The Chinese patent CN101830970 and CN102276696A are improved on the basis of CN1404487, CN101830970A adopts buffer solution to prepare a daptomycin crude product into a sample solution, then the sample solution is put on a composite YT-01 column to be adsorbed by a reverse phase silica gel material, and then aqueous solution of strong polar solvent is used as desorbent to perform gradient elution or constant concentration elution; CN102276696a is eluted after loading and binding semi-purified daptomycin to a gel-type weak anion resin. The two patent technologies simplify the flow and reduce the cost, but are difficult to realize industrialized production.
CN103224547A is prepared through regulating pH value of fermentation liquid, collecting mycelium liquid, extracting with butanol and dipotassium hydrogen phosphate solution repeatedly, concentrating the extractive liquid under reduced pressure, crystallizing to obtain crude daptomycin product, separating the crude daptomycin product with macroporous resin chromatography, crystallizing the desorption liquid with isopropanol, recrystallizing to obtain refined daptomycin product with purity up to 99.5%, but the process is complicated, uses a large amount of organic solvent, is unfavorable for environment protection, has low yield, and is unfavorable for industrial production.
CN102718839a discloses a method for separating and purifying daptomycin, which comprises the following steps: ceramic membrane filtration of daptomycin fermentation broth, macroporous adsorption resin chromatography, nanofiltration concentration and hydrophobic resin column chromatography for first step coarse separation; performing ion exchange resin column chromatography, performing second coarse separation by hydrophobic resin column chromatography, ultrafiltering, and freeze drying to obtain daptomycin with purity of more than 99.2%. The method has the defects of long operation steps and low yield.
In summary, the purification process of daptomycin has the defects of complicated operation, long steps, high production cost, low yield and purity and the like, so that the purification process of daptomycin with simple operation, short steps and high yield and purity is needed to be provided.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a purification method of daptomycin with high purity and high yield. The method overcomes the defects of the prior purification technology of the daptomycin, improves the purity of the daptomycin, shortens the purification process steps, and improves the yield of the daptomycin purification.
The technical scheme of the invention is as follows:
A method for purifying daptomycin, comprising the steps of:
(1) Filtering the daptomycin fermentation liquor by a ceramic membrane;
(2) Carrying out hydrophobic resin chromatography;
(3) Ion exchange resin chromatography;
(4) Desalting by hydrophobic resin chromatography and removing endotoxin;
(5) Nanofiltration and concentration;
(6) And (5) freeze drying.
Preferably, the daptomycin fermentation broth is filtered by a ceramic membrane in the step (1), the pore diameter of the ceramic membrane is 50-200nm, and the filtrate is collected.
Preferably, the hydrophobic resin of step (2) is an XR3SP resin or an XR30SP resin, preferably an XR3SP resin; the diameter-to-height ratio of the resin column is 1:3-4.
Preferably, the hydrophobic resin chromatography step in the step (2) comprises the steps of resin column balancing, loading the hydrophobic chromatography column and gradient flushing; the gradient flushing process is to flush with acetonitrile water solution containing sodium acetate after loading.
Further preferably, the loading amount of the step (2) is 8-15g/L resin, and the loading flow rate is 1-2 times of the bed volume/hour
Further preferably, the gradient rinsing process is as follows: washing with 15-20% acetonitrile water solution (containing 3-4 g/L of sodium acetate and having pH of 4.0-4.5), then washing with 15-20% acetonitrile water solution (containing 3-4 g/L of sodium acetate and having pH of 5.0-5.5), and finally eluting with 25-30% acetonitrile water solution (containing 3-4 g/L of sodium acetate and having pH of 5.5-6.0); and collecting elution effluent containing daptomycin.
Still further, the gradient rinse process flow rate is 0.5 to 2 bed volumes per hour.
Preferably, the ion exchange resin in step (3) is an NQ-M resin; the diameter-to-height ratio of the resin column is 1:3-4.
Preferably, the ion exchange resin chromatography process in the step (3) comprises a resin column balancing, loading the ion exchange resin column and a gradient flushing process; the gradient flushing process is to flush with sodium chloride solution containing sodium acetate after the sample loading is finished.
Further preferably, the resin loading amount in the step (3) is 6-12g/L, and the pH of the loading solution is 6.0-6.5; the loading flow rate is 1-3 times of the bed volume per hour.
Further preferably, the gradient rinsing process of step (3) is as follows: washing with 1.2-3 g/L sodium chloride solution (containing 1.5-2.0 g/L sodium acetate, pH 5.0-5.5), and eluting with 4-8 g/L sodium chloride solution (containing 1.5-2.0 g/L sodium acetate, pH 5.0-5.5); and collecting elution effluent containing daptomycin.
Further, the flow rate of the gradient flushing process in the step (3) is 1-3 times of the volume of the bed per hour.
Preferably, in the step (4), the collected liquid in the step (3) is subjected to desalting and endotoxin removal by using XR30SP resin; the diameter-to-height ratio of the resin column is 1:1-2.
Further preferably, the step (4) comprises the steps of adjusting the pH of the collection liquid obtained in the step (3) to 2.6-3.6, loading the collection liquid onto a hydrophobic resin column, washing the collection liquid with 10-20% ethanol aqueous solution after loading, and eluting the collection liquid with 75-85% ethanol aqueous solution. The loading flow rate is 2-5 times of the volume/hour of the bed, and the gradient flushing flow rate is 3-6 times of the volume/hour of the bed.
Preferably, the nanofiltration concentration process in the step (5) adopts a polyethersulfone nanofiltration membrane, and the molecular weight cut-off is 200-1000 daltons.
Further preferably, in the step (5), the collected liquid in the step (4) is diluted by 2-4 times by water for injection, the pH value is regulated to 2.6-4.6, the concentrated liquid is washed by the water for injection in the nanofiltration process until the ethanol content in the concentrated liquid is lower than 0.05%, the nanofiltration concentration is continued, and the nanofiltration is stopped when the daptomycin content in the concentrated liquid is higher than 90000 mg/L.
Preferably, the freeze drying process in the step (6) is that the freezing is carried out for 2 to 8 hours at the temperature of minus 60 ℃ to minus 45 ℃ and the pressure of lower than 20 Pa; then freeze-drying for 50-80 h at-60-20 ℃ and 20-60 pa; finally, freeze-drying is carried out for 2-8 h under the conditions of 20-30 ℃ and the pressure lower than 10 pa.
The purification method of daptomycin provided by the invention has the advantages of simple operation, short steps, short production period, high product yield and high purity of daptomycin, and is beneficial to large-scale production.
Detailed Description
The present invention will be clearly illustrated by the following specific examples, which are to be understood for illustrative purposes only and are not intended to limit the scope of the present invention.
The sources of fermentation broth used in the examples below can be referred to prior art methods for preparing daptomycin fermentation broth. Other materials, starting materials, reagents are commercially available unless otherwise specified.
Example 1
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the daptomycin content is 1.6 g/L), 3L of ethanol is added, the solution is filtered by a ceramic membrane with the thickness of 0.2 mu m at room temperature, when the volume of the concentrated solution is less than 10L, purified water is added for washing, and when HPLC (high performance liquid chromatography) detects that the daptomycin content in the ceramic membrane permeate is less than 50mg/L, the filtration is stopped, 70.1L of permeate is collected together, and 43.6g of daptomycin is contained.
2) Hydrophobic resin chromatography
The permeate obtained by ceramic membrane filtration was loaded onto an XR3SP resin column (resin usage 5L, diameter-to-height ratio 1:3), the loading flow rate was 1 time the bed volume/hour, after the loading was completed, 3BV was washed successively with a 20% acetonitrile solution (pH 4.5, containing sodium acetate 3.5 g/L), 8BV was washed again with a 20% acetonitrile solution (pH 5.5, containing sodium acetate 3.5 g/L), eluted again with a 25% acetonitrile solution (pH 6.0, containing sodium acetate 3.5 g/L), collection was started after the on-line ultraviolet monitoring of the absorption peak, the total collection eluate was 14.6L, the HPLC detection purity was 91.8%, and daptomycin was 34.8g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH to 6.5, and loading the diluted liquid into an NM-Q resin column (resin dosage is 4L, resin column diameter-to-height ratio is 1:3), wherein the flow rate is 2 times of the volume of the column bed per hour. Eluting with 26BV 1.7g/L sodium chloride solution (containing 1.6g/L sodium acetate and pH5.5 adjusted by acetic acid), eluting with 7g/L sodium chloride solution (containing 1.6g/L sodium acetate and pH 5.5) at a flow rate of 2 times the volume of the bed/hr, collecting after on-line ultraviolet monitoring absorption peak, collecting eluate 8.9L altogether, and detecting by HPLC the purity of 99.8% and containing daptomycin 27.1g.
4) Desalting and endotoxin removing by hydrophobic resin chromatography
The pH of the collection obtained in step 3) was adjusted to 3.2 and the sample was applied to an XR30SP resin column (resin dose 0.6L, aspect ratio 1: 2) The flow rate was 3BV/h. The resin column is washed by 4BV 10% ethanol solution, then eluted by 75% ethanol solution, after the absorption peak is monitored by online ultraviolet, the collection is started, when the effluent becomes colorless, the collection is stopped, 650ml of the eluent is collected altogether, the purity of HPLC detection is 99.8%, the daptomycin content is 26.0g, and the endotoxin content detected by a limulus reagent method is less than 0.06EU/mg.
5) Nanofiltration concentration
Diluting the collected liquid in the step 4) to 1.5L by adding water for injection, regulating the pH value to 3.8 by using hydrochloric acid, carrying out nanofiltration by using a polyethersulfone nanofiltration membrane with a molecular weight cut-off of 200 daltons, washing the nanofiltration concentrated solution by using water for injection in the nanofiltration process until the ethanol content of the nanofiltration permeate is lower than 0.05%, continuing the nanofiltration after the washing is finished until the daptomycin content in the nanofiltration concentrated solution is higher than 90000mg/L, stopping the nanofiltration, and collecting 280ml of nanofiltration concentrated solution.
6) Freeze drying
Lyophilizing the nanofiltration concentrate obtained in step 5) under the following conditions: freezing for 2-8 hours under the condition that the pressure is lower than 20Pa at minus 60 ℃ to minus 45 ℃; then freeze-drying for 50-80 hours under the conditions of minus 60 ℃ to 20 ℃ and 20-60 Pa; finally, the mixture is freeze-dried for 2 to 8 hours at the temperature of 20 to 30 ℃ and the pressure of less than 10Pa, and 23.9g of the tobramycin freeze-dried powder is obtained, and the HPLC purity is 99.7 percent.
Example 2
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the daptomycin content is 1.6 g/L), 3.5L of ethanol is added, the solution is filtered by a 50nm ceramic membrane at room temperature, when the volume of the concentrated solution is less than 10L of the volume of the fermentation liquor, purified water is added to the original volume, when HPLC (high performance liquid chromatography) detects that the daptomycin content in the ceramic membrane permeate is less than 50mg/L, the filtration is stopped, and 79.3L of permeate is collected altogether, and 43.8g of daptomycin is contained.
2) Hydrophobic resin chromatography
The permeate obtained by ceramic membrane filtration was loaded onto an XR3SP resin column (resin usage 6L, diameter-to-height ratio 1:4), the loading flow rate was 2 times the bed volume/hour, after loading was completed, 3BV was washed successively with 15% acetonitrile solution (pH 4.0, containing sodium acetate 4.0 g/L), 8BV was washed again with 15% acetonitrile solution (pH 5.0, containing sodium acetate 4.0 g/L), eluted with 30% acetonitrile solution (pH 5.5, containing sodium acetate 4.0 g/L), collection was started after on-line ultraviolet monitoring of the absorption peak, the total eluent was collected 15.3L, HPLC detection purity was 90.6%, and daptomycin was 33.2g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, and loading the diluted liquid into an NM-Q resin column (resin dosage is 5.5L, the diameter-to-height ratio of the resin column is 1:4), wherein the flow rate is 1 times of the volume of the bed per hour. The impurities were washed with 30BV 1.2g/L sodium chloride solution (containing 2g/L sodium acetate, pH5.0 adjusted with acetic acid), then eluted with 4g/L sodium chloride solution (containing 2g/L sodium acetate, pH 5.0) at a flow rate of 3 times the bed volume/hour, and collected after on-line UV monitoring absorption peak, 9.7L of the eluate was collected altogether, and the HPLC detection purity was 99.6%, containing 24.9g of daptomycin.
4) Desalting and endotoxin removing by hydrophobic resin chromatography
The pH of the collection obtained in step 3) was adjusted to 2.6 and the sample was applied to an XR30SP resin column (resin volume 450mL, aspect ratio 1: 2) The flow rate was 2BV/h. The resin column is washed by 4BV 20% ethanol solution, then eluted by 85% ethanol solution, after the absorption peak is monitored by online ultraviolet, the collection is started, when the effluent becomes colorless, the collection is stopped, 500mL of the total collected eluent is collected, the purity of HPLC detection is 99.7%, the daptomycin content is 23.8g, and the endotoxin content detected by a limulus reagent method is less than 0.06EU/mg.
5) Nanofiltration concentration
Diluting the collected liquid in the step 4) to 2.0L by adding water for injection, regulating the pH value to 4.6 by using hydrochloric acid, carrying out nanofiltration by using a polyethersulfone nanofiltration membrane with the molecular weight cut-off of 1000 daltons, washing the nanofiltration concentrated solution by using water for injection in the nanofiltration process until the ethanol content of the nanofiltration permeate is lower than 0.05%, continuing the nanofiltration after the washing is finished until the daptomycin content in the nanofiltration concentrated solution is higher than 90000mg/L, stopping the nanofiltration, and collecting 250ml of nanofiltration concentrated solution.
6) Freeze drying
Lyophilizing the nanofiltration concentrate obtained in step 5) under the following conditions: freezing for 2-8 hours under the condition that the pressure is lower than 20Pa at minus 60 ℃ to minus 45 ℃; then freeze-drying for 50-80 hours under the conditions of minus 60 ℃ to 20 ℃ and 20-60 Pa; finally, the mixture is freeze-dried for 2 to 8 hours at the temperature of 20 to 30 ℃ and the pressure of less than 10Pa, and the mixture reaches 21.5g of the tobramycin freeze-dried powder, and the HPLC purity is 99.5 percent.
Example 3
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the daptomycin content is 1.6 g/L), 2.5L of ethanol is added, the solution is filtered by a 0.1 mu m ceramic membrane at room temperature, when the volume of the concentrated solution is less than 10L of the volume of the fermentation liquor, purified water is added to the original volume, and when the HPLC detection of the daptomycin content in the ceramic membrane permeate is less than 50mg/L, the filtration is stopped, and 84L of permeate is collected together, wherein 43.7g of daptomycin is contained.
2) Hydrophobic resin chromatography
The permeate obtained by ceramic membrane filtration was loaded onto an XR30SP resin column (resin usage 3.5L, diameter-to-height ratio 1:3), the loading flow rate was 1 time the bed volume/hour, after the loading was completed, 4BV was washed successively with 15% acetonitrile solution (pH 4.5, containing sodium acetate 3.0 g/L), 9BV was washed again with 15% acetonitrile solution (pH 5.5, containing sodium acetate 3.0 g/L), eluted with 30% acetonitrile solution (pH 6.0, containing sodium acetate 3.0 g/L), collection was started after the on-line ultraviolet monitoring absorption peak, the total collected eluate was 15.2L, HPLC detection purity was 90.9%, containing daptomycin 32.5g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH to 6.5, and loading the diluted liquid into an NM-Q resin column (resin dosage is 3L, resin column diameter-to-height ratio is 1:3), wherein the flow rate is 3 times of the volume of the column bed per hour. 20BV 2.0g/L sodium chloride solution (containing 1.5g/L sodium acetate, pH5.5 adjusted by acetic acid) is used for washing impurities, then 8g/L sodium chloride solution (containing 1.5g/L sodium acetate, pH 5.5) is used for eluting, the flow rate is 1 time of the volume of a column bed per hour, the collection is started after the absorption peak is monitored by on-line ultraviolet, 7.2L of eluent is collected together, the purity of the eluent is 99.5 percent by HPLC detection, and 24.3g of daptomycin is contained.
4) Desalting and endotoxin removing by hydrophobic resin chromatography
The pH of the collection obtained in step 3) was adjusted to 3.6 and the sample was applied to an XR30SP resin column (resin volume 500mL, aspect ratio 1: 2) The flow rate was 5BV/h. The resin column is washed by 4BV 10% ethanol solution, then is eluted by 75% ethanol solution, the washing and eluting flow rate is 6BV/h, after the absorption peak is monitored by on-line ultraviolet, the collection is started, when the effluent becomes colorless, the collection is stopped, 500mL of eluent is collected altogether, the HPLC detection purity is 99.6%, the daptomycin content is 23.2g, and the endotoxin content detected by a limulus reagent method is less than 0.06EU/mg.
5) Nanofiltration concentration
Adding the eluent collected in the step 4) into water for injection to dilute to 1.5L, adjusting the pH value to 4.6 by hydrochloric acid, carrying out nanofiltration by using a polyethersulfone nanofiltration membrane with the molecular weight cut-off of 500 daltons, adding water for injection to wash nanofiltration concentrated solution in the nanofiltration process until the ethanol content of nanofiltration permeate is lower than 0.05%, continuing nanofiltration until the content of daptomycin in the nanofiltration concentrated solution is higher than 90000mg/L after the washing is finished, stopping nanofiltration, and collecting 240ml of nanofiltration concentrated solution.
6) Freeze drying
Lyophilizing the nanofiltration concentrate obtained in step 5) under the following conditions: freezing for 2-8 hours under the condition that the pressure is lower than 20Pa at minus 60 ℃ to minus 45 ℃; then freeze-drying for 50-80 hours under the conditions of minus 60 ℃ to 20 ℃ and 20-60 Pa; finally, the mixture is freeze-dried for 2 to 8 hours at the temperature of 20 to 30 ℃ and the pressure of less than 10Pa, and the final product reaches 21.4g of the tobramycin freeze-dried powder, and the HPLC purity is 99.5 percent.
Example 4
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the daptomycin content is 1.6 g/L), 3L of ethanol is added, the solution is filtered by a ceramic membrane with the thickness of 0.2 mu m at room temperature, when the volume of the concentrated solution is less than 10L of the volume of the fermentation liquor, purified water is added to the original volume, and when the HPLC detection of the daptomycin content in the ceramic membrane permeate is less than 50mg/L, the filtration is stopped, 71.5L of permeate is collected together, and 43.2g of daptomycin is contained.
2) Hydrophobic resin chromatography
The permeate obtained by ceramic membrane filtration was loaded onto an XR30SP resin column (resin usage 5L, diameter-to-height ratio 1:3), the loading flow rate was 1 time the bed volume/hour, after the loading was completed, 3BV was washed successively with a 20% acetonitrile solution (pH 4.5, containing sodium acetate 3.5 g/L), 8BV was washed again with a 20% acetonitrile solution (pH 5.5, containing sodium acetate 3.5 g/L), eluted again with a 25% acetonitrile solution (pH 6.0, containing sodium acetate 3.5 g/L), collection was started after the on-line ultraviolet monitoring of the absorption peak, the total collection eluate was 14.9L, the HPLC detection purity was 91.3%, and daptomycin was 34.0g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH to 6.5, and loading the diluted liquid into an NM-Q resin column (resin dosage is 4L, resin column diameter-to-height ratio is 1:3), wherein the flow rate is 2 times of the volume of the column bed per hour. Eluting with 26BV 1.75g/L sodium chloride solution (containing 1.64g/L sodium acetate, pH5.5 adjusted with acetic acid), eluting with 7g/L sodium chloride solution (containing 1.64g/L sodium acetate, pH 5.5) at a flow rate of 2 times the volume of the bed/hr, collecting after on-line ultraviolet monitoring absorption peak, collecting eluate 8.6L altogether, and detecting with HPLC to have purity of 99.6% and daptomycin content of 26.5g.
4) Desalting and endotoxin removing by hydrophobic resin chromatography
The pH of the collection obtained in step 3) was adjusted to 3.2 and the sample was applied to an XR30SP resin column (resin volume 500mL, aspect ratio 1: 1) The flow rate was 3BV/h. The resin column is washed by 4BV 10% ethanol solution, then eluted by 75% ethanol solution, after the absorption peak is monitored by online ultraviolet, the collection is started, when the effluent becomes colorless, the collection is stopped, 500mL of the total collected eluent is collected, the purity of HPLC detection is 99.6%, the daptomycin content is 25.2g, and the endotoxin content detected by a limulus reagent method is less than 0.06EU/mg.
5) Nanofiltration concentration
Diluting the collected liquid in the step 4) to 1.5L by adding water for injection, regulating the pH value to 3.8 by using hydrochloric acid, carrying out nanofiltration by using a polyethersulfone nanofiltration membrane with a molecular weight cut-off of 200 daltons, adding water for injection to wash nanofiltration concentrated solution in the nanofiltration process until the ethanol content of nanofiltration permeation solution is lower than 0.05%, continuing nanofiltration after washing until the content of daptomycin in the nanofiltration concentrated solution is higher than 90000mg/L, stopping nanofiltration, and collecting 265ml of nanofiltration concentrated solution
6) Freeze drying
Lyophilizing the nanofiltration concentrate obtained in step 5) under the following conditions: freezing for 2-8 hours under the condition that the pressure is lower than 20Pa at minus 60 ℃ to minus 45 ℃; then freeze-drying for 50-80 hours under the conditions of minus 60 ℃ to 20 ℃ and 20-60 Pa; finally, the mixture is freeze-dried for 2 to 8 hours at the temperature of 20 to 30 ℃ and the pressure of less than 10Pa, and 23.1g of the tobramycin freeze-dried powder is obtained, and the HPLC purity is 99.5 percent.
Example 5
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the daptomycin content is 1.6 g/L), 3L of ethanol is added, the solution is filtered by a ceramic membrane with the thickness of 0.2 mu m at room temperature, when the volume of the concentrated solution is less than 10L of the volume of the fermentation liquor, purified water is added to the original volume, and when the HPLC detection of the daptomycin content in the ceramic membrane permeate is less than 50mg/L, the filtration is stopped, 70.2L of permeate is collected altogether, and 43.3g of daptomycin is contained.
2) Hydrophobic resin chromatography
The permeate obtained by ceramic membrane filtration is loaded on a chromatographic resin column III (resin dosage is 5L, diameter-to-height ratio is 1:3), the loading flow rate is 1 time of column bed volume/hour, after loading is finished, 3BV is washed by 20% acetonitrile solution (pH 4.5, containing sodium acetate 3.5 g/L), 8BV is washed by 20% acetonitrile solution (pH 5.5, containing sodium acetate 3.5 g/L), eluting by 25% acetonitrile solution (pH 6.0, containing sodium acetate 3.5 g/L), collection is started after an online ultraviolet monitoring absorption peak, eluent is collected altogether 15.2L, HPLC detection purity is 89.1%, and daptomycin content is 31.6g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH to 6.5, and loading the diluted liquid into FPDA-13 resin columns (the resin dosage is 4L, the diameter-to-height ratio of the resin columns is 1:3), wherein the flow rate is 2 times of the volume of the column bed per hour. Eluting with 26BV 1.75g/L sodium chloride solution (containing 1.64g/L sodium acetate, pH 5.5 adjusted with acetic acid), eluting with 7g/L sodium chloride solution (containing 1.64g/L sodium acetate, pH 5.5) at a flow rate of 2 times the volume of the bed/hr, collecting after on-line ultraviolet monitoring absorption peak, collecting eluate 8.1L altogether, and detecting with HPLC to have purity of 99.3% and daptomycin content of 23.7g.
4) Desalting and endotoxin removing by hydrophobic resin chromatography
The pH of the collection obtained in step 3) was adjusted to 3.2, and the mixture was applied to an HP20SS resin column (resin amount 500mL, aspect ratio 1: 1) The flow rate was 3BV/h. The resin column is washed by 4BV 10% ethanol solution, then eluted by 75% ethanol solution, after the absorption peak is monitored by online ultraviolet, the collection is started, when the effluent becomes colorless, the collection is stopped, 50mL of the eluent is collected altogether, the purity of HPLC detection is 99.2%, the daptomycin content is 21.7g, and the endotoxin content detected by a limulus reagent method is less than 0.3EU/mg.
5) Nanofiltration concentration
Diluting the collected liquid in the step 4) to 1.5L by adding water for injection, regulating the pH value to 3.8 by using hydrochloric acid, carrying out nanofiltration by using a polyethersulfone nanofiltration membrane with a molecular weight cut-off of 200 daltons, washing the nanofiltration concentrated solution by using water for injection in the nanofiltration process until the ethanol content of the nanofiltration permeate is lower than 0.05%, continuing the nanofiltration after the washing is finished until the daptomycin content in the nanofiltration concentrated solution is higher than 90000mg/L, stopping the nanofiltration, and collecting 230ml of nanofiltration concentrated solution.
6) Freeze drying
Lyophilizing the nanofiltration concentrate obtained in step 5) under the following conditions: freezing for 2-8 hours under the condition that the pressure is lower than 20Pa at minus 60 ℃ to minus 45 ℃; then freeze-drying for 50-80 hours under the conditions of minus 60 ℃ to 20 ℃ and 20-60 Pa; finally, the mixture is freeze-dried for 2 to 8 hours at the temperature of 20 to 30 ℃ and the pressure of less than 10Pa, and the final product reaches 19.5g of the tobramycin freeze-dried powder, and the HPLC purity is 99.3 percent.
Example 6
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the daptomycin content is 1.6 g/L), 3L of ethanol is added, the solution is filtered by a ceramic membrane with the thickness of 0.2 mu m at room temperature, when the volume of the concentrated solution is less than 10L of the volume of the fermentation liquor, purified water is added to the original volume, and when the HPLC detection of the daptomycin content in the ceramic membrane permeate is less than 100mg/L, the filtration is stopped, 70.1L of permeate is collected together, and 43.4g of daptomycin is contained.
2) Hydrophobic resin chromatography
The permeate obtained by ceramic membrane filtration was loaded onto an XR30SP resin column (resin usage 5L, diameter-to-height ratio 1:3), the loading flow rate was 1 time the bed volume/hour, after loading was completed, 10BV was washed with 33% methanol (pH 5.5, containing sodium acetate 2.5 g/L), eluted with 50% methanol (pH 6.5, containing sodium acetate 2.5 g/L), and after on-line UV monitoring the absorption peak, collection was started, 10.4L of the eluate was collected altogether, HPLC detection purity was 89.4%, and daptomycin was 34.6g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 20L of purified water, adjusting the pH to 6.5, and loading the diluted liquid into an NM-Q resin column (resin dosage is 4L, resin column diameter-to-height ratio is 1:3), wherein the flow rate is 2 times of the volume of a column bed per hour. Eluting with 5.8g/L sodium chloride solution (containing 2.5g/L sodium acetate) at a flow rate of 2 times the volume of the bed/hr, collecting after on-line ultraviolet monitoring absorption peak, collecting 7.2L eluate, and detecting with HPLC to have purity of 99.0% and daptomycin content of 23.3g.
4) Desalting and endotoxin removing by hydrophobic resin chromatography
The collection solution obtained in step 3) was adjusted to pH3.2 and loaded onto a HZ016 resin column (resin usage 500mL, aspect ratio 1: 1) The flow rate was 3BV/h. Washing the resin column with 4BV 10% ethanol solution, eluting with 75% ethanol solution, monitoring absorption peak by online ultraviolet, collecting, stopping collecting when effluent becomes colorless, collecting 600L eluate, and detecting purity by HPLC to 99.1%, wherein daptomycin content is 22.1g, and detecting endotoxin content by limulus reagent method is less than 0.3EU/mg.
5) Nanofiltration concentration
Diluting the collected liquid in the step 4) to 1.5L by adding water for injection, regulating the pH value to 3.8 by using hydrochloric acid, carrying out nanofiltration by using a polyethersulfone nanofiltration membrane with a molecular weight cut-off of 200 daltons, washing the nanofiltration concentrated solution by using water for injection in the nanofiltration process until the ethanol content of the nanofiltration permeate is lower than 0.05%, continuing the nanofiltration after the washing is finished until the daptomycin content in the nanofiltration concentrated solution is higher than 90000mg/L, stopping the nanofiltration, and collecting 250ml of nanofiltration concentrated solution.
6) Freeze drying
Lyophilizing the nanofiltration concentrate obtained in step 5) under the following conditions: freezing for 2-8 hours under the condition that the pressure is lower than 20Pa at minus 60 ℃ to minus 45 ℃; then freeze-drying for 50-80 hours under the conditions of minus 60 ℃ to 20 ℃ and 20-60 Pa; finally, the mixture is freeze-dried for 2 to 8 hours at the temperature of 20 to 30 ℃ and the pressure of less than 10Pa, and the final product reaches to 20.1g of the tobramycin freeze-dried powder, and the HPLC purity is 99.0 percent.
Comparative example 1
Cooling 30L of daptomycin fermentation broth (daptomycin content is 1.6 g/L) to room temperature, clarifying with a ceramic membrane of 0.2 μm, continuously adding purified water for washing in the filtering process, discarding bacterial residues, and collecting 89L of clarified liquid;
Purifying the clarified solution by a 5L HP20 resin column, eluting with 33% methanol containing 30mM sodium acetate at pH5.5 and 50% methanol containing 30mM sodium acetate at pH6.5, and collecting daptomycin eluate with a purity of > 92% as 9L;
Diluting the collected liquid 9L with purified water until the concentration is 10% of methanol, purifying by 5LHP20SS resin, eluting with methanol with pH5.5 containing 30mM sodium acetate and 45% methanol with pH6.5 containing 30mM sodium acetate to obtain daptomycin eluting collection liquid 8L with purity of more than 94%;
Purifying the collected liquid 8L by FPDA-13 ion exchange resin, removing the organic solvent, eluting by using 1M sodium chloride and 30mM sodium acetate to obtain daptomycin eluting collected liquid 7L with the purity of more than 96%;
Regulating pH value of the collected feed liquid to 6.0, concentrating by a nanofiltration membrane system with molecular weight cut-off of 360 daltons, and removing small molecular impurities to obtain 500ml of tobramycin concentrate; removing endotoxin from the concentrated solution by an ultrafiltration membrane system with a molecular weight cutoff of 10KD, collecting the permeate, and detecting the endotoxin content of less than 0.3EU/mg by a limulus reagent method; concentrating the permeate to 60g/L, regulating the pH to 6.0, adding isopropanol for precipitation, and carrying out centrifugal filtration to collect the precipitate;
After the collected precipitate was dissolved in 10L of water, the pH was adjusted to 5.0, and after desalting with a cationic resin HZ016, the effluent was collected. The obtained effluent is concentrated by nanofiltration, sterilized and filtered by a 0.22 mu m membrane, and then is filled, frozen and preserved at the temperature of minus 20 ℃ to obtain 16.8g of daptomycin frozen block, and the HPLC purity of the daptomycin is 98.3 percent.
Comparative example 2
The daptomycin fermentation liquor (30L, daptomycin content is 1.6 g/L) is regulated to pH3.0 by phosphoric acid, macromolecular substances such as water-soluble proteins, pigments and the like in the daptomycin fermentation liquor are removed by ceramic membrane filtration with the aperture of 0.05 mu m, then the ceramic membrane is circularly washed (discarded) by 1-2 times of volume of phosphoric acid aqueous solution with the pH of 3.0, and then the ceramic membrane is circularly washed by 4-5 times of volume of sodium hydroxide solution with the pH of 9.0 to obtain ceramic membrane filtrate, and the obtained filtrate is clear and transparent and has the color of reddish yellow.
When the pH value of the filtrate is regulated to 6.0 by dilute acetic acid, the filtrate is subjected to primary passing through small-section D101 resin columns (the diameter-to-height ratio is 1:10), the serial connection is disconnected, each small Duan Zhu is respectively washed by water until each effluent is basically colorless, and then 3 small-section D101 resin columns are sequentially connected in series and then are connected in series with another D101 resin column (the diameter-to-height ratio is 1:10); and eluted with 0.06N sodium acetate, 30% ethanol solution at pH 6.5.
The resulting eluate was subjected to pH5.5 adjustment with dilute acetic acid and then to D301 resin column (diameter-to-height ratio 1:10), and the resin column was washed with water and then eluted with 300mM sodium chloride solution having pH5.5 to 6.0. The eluted material of the D301 resin column is concentrated by nanofiltration membrane with the molecular weight cut-off of 100-200, and the concentration of the daptomycin is 20% (w/w) after concentration. The concentrated solution is lyophilized in vacuum to obtain 18.4g of daptomycin lyophilized powder with an HPLC purity of 97.5%.
Claims (1)
1. A method for purifying daptomycin, comprising the steps of:
(1) Filtering the daptomycin fermentation liquor by a ceramic membrane;
(2) XR3SP or XR30SP hydrophobic resin chromatography;
Balancing a resin column, loading a sample, and after the loading is finished, washing with acetonitrile aqueous solution containing sodium acetate in a gradient manner; washing with 15-20% acetonitrile water solution with pH of 4.0-4.5 and 3-4 g/L sodium acetate, then washing with 15-20% acetonitrile water solution with pH of 5.0-5.5 and 3-4 g/L sodium acetate, and finally eluting with 25-30% acetonitrile water solution with pH of 5.5-6.0 and 3-4 g/L sodium acetate; collecting elution effluent containing daptomycin;
(3) NM-Q ion exchange resin chromatography;
Balancing a resin column, loading a sample, and after the loading is finished, washing with sodium chloride solution containing sodium acetate in a gradient manner; washing with 1.2-3 g/L sodium chloride solution with pH of 5.0-5.5 and 1.5-2.0 g/L sodium acetate, and eluting with 4-8 g/L sodium chloride solution with pH of 5.0-5.5 and 1.5-2.0 g/L sodium acetate; collecting elution effluent containing daptomycin;
(4) Removing salt and endotoxin by XR30SP hydrophobic resin chromatography;
adjusting the pH value of the collection liquid obtained in the step (3) to 2.6-3.6, loading the collection liquid on a sample hydrophobic resin column, washing the collection liquid with 10-20% ethanol solution after loading, eluting the collection liquid with 75-85% ethanol solution, and collecting elution effluent liquid containing daptomycin;
(5) Nanofiltration and concentration of a polyether sulfone nanofiltration membrane with the molecular weight cut-off of 200-1000 daltons;
Diluting the collected liquid in the step (4) by 2-4 times with water for injection, adjusting the pH value to 2.6-4.6, washing the concentrated liquid with water for injection in the nanofiltration process until the ethanol content in the concentrated liquid is lower than 0.05%, continuing nanofiltration concentration, and stopping nanofiltration when the daptomycin content in the concentrated liquid is higher than 90000 mg/L;
(6) Freeze drying;
freezing for 2-8 hours at the temperature of minus 60 ℃ to minus 45 ℃ and the pressure of lower than 20 Pa; then at again
Freeze-drying at-60-20 deg.c and pressure of 20-60 pa for 50-80 hr; finally, freeze-drying is carried out for 2-8 h under the conditions of 20-30 ℃ and the pressure lower than 10 pa.
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