CN113004373A - Daptomycin purification method - Google Patents
Daptomycin purification method Download PDFInfo
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- CN113004373A CN113004373A CN201911317729.0A CN201911317729A CN113004373A CN 113004373 A CN113004373 A CN 113004373A CN 201911317729 A CN201911317729 A CN 201911317729A CN 113004373 A CN113004373 A CN 113004373A
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- daptomycin
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- sodium acetate
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- nanofiltration
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- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 117
- 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 117
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 116
- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000000746 purification Methods 0.000 title claims abstract description 13
- 239000011347 resin Substances 0.000 claims abstract description 111
- 229920005989 resin Polymers 0.000 claims abstract description 111
- 239000012528 membrane Substances 0.000 claims abstract description 42
- 239000000919 ceramic Substances 0.000 claims abstract description 35
- 238000004108 freeze drying Methods 0.000 claims abstract description 31
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 31
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 29
- 238000001914 filtration Methods 0.000 claims abstract description 28
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- 239000002158 endotoxin Substances 0.000 claims abstract description 18
- 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 16
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 16
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 16
- 150000003839 salts Chemical class 0.000 claims abstract description 3
- 238000001728 nano-filtration Methods 0.000 claims description 73
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 60
- 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
- 238000011068 loading method Methods 0.000 claims description 44
- 238000005406 washing Methods 0.000 claims description 41
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 38
- 239000011780 sodium chloride Substances 0.000 claims description 19
- 238000010828 elution Methods 0.000 claims description 11
- 239000004695 Polyether sulfone Substances 0.000 claims description 8
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 8
- 229920006393 polyether sulfone Polymers 0.000 claims description 8
- 238000011067 equilibration Methods 0.000 claims description 4
- 238000011210 chromatographic step Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 88
- 239000007788 liquid Substances 0.000 description 67
- 238000004128 high performance liquid chromatography Methods 0.000 description 47
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 31
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 239000003480 eluent Substances 0.000 description 17
- 238000012544 monitoring process Methods 0.000 description 17
- 239000008213 purified water Substances 0.000 description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- 239000008215 water for injection Substances 0.000 description 12
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 8
- 238000007865 diluting Methods 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 239000012466 permeate Substances 0.000 description 8
- 241000239218 Limulus Species 0.000 description 7
- 238000011033 desalting Methods 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000007710 freezing Methods 0.000 description 6
- 230000008014 freezing Effects 0.000 description 6
- 238000005374 membrane filtration Methods 0.000 description 6
- 238000011010 flushing procedure Methods 0.000 description 5
- 239000012535 impurity 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
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000000149 penetrating effect Effects 0.000 description 4
- 108010028921 Lipopeptides Proteins 0.000 description 3
- 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
- 235000010633 broth Nutrition 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229960003085 meticillin Drugs 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000234435 Lilium 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
- 230000007547 defect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 229940090044 injection Drugs 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 229960003165 vancomycin Drugs 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
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) 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)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 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
- 241000194032 Enterococcus faecalis Species 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
- 229940121657 clinical drug Drugs 0.000 description 1
- 150000001875 compounds Chemical class 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
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 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
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000005070 sampling Methods 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
- 238000009777 vacuum freeze-drying 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
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- 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 daptomycin purification method. The method comprises the following steps: 1) filtering the daptomycin fermentation liquor by using a ceramic membrane; 2) carrying out hydrophobic resin chromatography; 3) ion exchange resin chromatography; 4) separating out salt and endotoxin by using a hydrophobic resin layer; 5) nano-filtering and concentrating; 6) and (5) freeze drying. The daptomycin purification method provided by the invention is simple to operate, short in steps, short in production period, high in product yield, high in daptomycin purity and beneficial to large-scale production.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a daptomycin purification method.
Background
Daptomycin is a cyclic lipopeptide material extracted from Streptomyces roseosporus fermentation broth, originally developed by Lily corporation in the United states in the 80 s, 11/1997, where Lily corporation assigned the worldwide exclusive development, production and marketing rights of Daptomycin to Cubist pharmaceutical company, 9/12/2003, and was first approved by FDA for the marketing of Cubicin (Daptomycin injection). It is the first cyclic lipopeptide antibiotic drug approved for the market and is used for treating complicated skin and skin structure infection caused by some gram-positive sensitive strains. Daptomycin was marketed in australia, germany, the netherlands, spain, the uk and in switzerland in 2007. Subsequently, they were registered in 10 countries such as Canada, Korea, European Union, etc. Us FDA re-approval at 3 months 2006; a novel indication for daptomycin is for the treatment of methicillin-sensitive Staphylococcus aureus (MSSA) or methicillin-resistant Staphylococcus aureus (MRSA) bacteremia and right endocarditis caused thereby.
Linezolid, developed by Pfizer in 2000, is one of only two new antibiotics developed in the last four decades, and cross-drug resistant bacteria have been reported in the market in less than a year. Daptomycin, however, has several unique properties that can confer clinical advantage. Firstly, the action mechanism of daptomycin is different from that of various antibacterial drugs on the market, which means that daptomycin cannot be influenced by cross drug resistance caused by other antibiotics, and the daptomycin serving as the first cyclic lipopeptide antibiotic applied to clinic has no report of cross drug-resistant bacteria 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 enterococcus faecalis, and vancomycin-resistant staphylococcus aureus. Third, daptomycin requires only 1 administration a day, significantly simplifying the dosage regimen and effectively reducing the cost of medical care compared to conventional antibiotics administered at least 2 times a day. Based on the factors, the medicine has wide market prospect in marketing.
At present, some daptomycin purification processes are reported at home and abroad. Chinese patent CN1404487 applied by the pharmaceutical company of Carbeter, USA, discloses a method for purifying daptomycin by using anion resin, which alternately uses anion resin FP-DA13, macroporous resin HP-20SS, anion resin Poros D50 or Poros 150 to separate and purify daptomycin, thus obtaining a daptomycin product with the purity of 98 percent, but the process is complicated and the cost is high, and the industrial scale production is difficult to realize.
Chinese patents CN101830970 and CN102276696A have been improved on the basis of CN1404487, CN101830970A is a sample solution prepared from a crude daptomycin product by using a buffer solution, and then the sample solution is loaded on a compound YT-01 column and adsorbed by a reversed phase silica gel material column, and then gradient elution or constant concentration elution is performed by using an aqueous solution of a strong polar solvent as a desorbent; CN102276696A is eluted after loading and binding semi-purified daptomycin to the gel type weak anion resin. Although the two patent technologies simplify the process and reduce the cost, the industrial production is difficult to realize.
CN103224547A firstly adjusts the pH of the daptomycin fermentation liquid, passes through a plate frame to collect mycelium liquid, the mycelium liquid is repeatedly extracted by butanol and dipotassium hydrogen phosphate solution, the extract liquid is decompressed, concentrated and crystallized to obtain a crude daptomycin product, the crude product is chromatographically separated by macroporous resin, and the desorption liquid is crystallized and recrystallized by isopropanol to obtain a refined daptomycin product, the purity reaches 99.5 percent, but the process is complicated, a large amount of organic solvent is used, the process is not environment-friendly, the yield is low, and the process is not beneficial to industrial production.
CN102718839A discloses a method for separating and purifying daptomycin, which comprises the following steps: filtering daptomycin fermentation liquor by using a ceramic membrane, performing macroporous adsorption resin chromatography, performing nanofiltration concentration, and performing hydrophobic resin column chromatography for first-step coarse separation; and (3) performing ion exchange resin column chromatography, performing hydrophobic resin column chromatography, performing second-step coarse separation, performing ultrafiltration, and performing freeze drying to obtain the daptomycin with the purity of more than 99.2%. The method has the disadvantages of long operation steps and low yield.
In conclusion, the daptomycin purification process has the defects of complicated operation, long steps, high production cost, low yield and purity and the like, so that the daptomycin purification process with simple operation, short steps and high yield and purity is urgently needed.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a method for purifying daptomycin with high purity and high yield. The method overcomes the defects of the prior daptomycin purification technology, improves the purity of daptomycin, shortens the purification process steps and improves the purification yield of daptomycin.
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 using a ceramic membrane;
(2) carrying out hydrophobic resin chromatography;
(3) ion exchange resin chromatography;
(4) separating out salt and endotoxin by using a hydrophobic resin layer;
(5) nano-filtering and concentrating;
(6) and (5) freeze drying.
Preferably, filtering the daptomycin fermentation liquor by using the ceramic membrane in the step (1), wherein the pore diameter of the ceramic membrane is 50-200nm, and collecting the filtrate.
Preferably, the hydrophobic resin of step (2) is an XR3SP resin or an XR30SP resin, preferably an XR3SP resin; the ratio of the diameter to the height of the resin column is 1: 3-4.
Preferably, the step (2) of hydrophobic resin chromatography comprises resin column equilibration, loading of a hydrophobic chromatography column and gradient washing; the gradient washing process refers to the gradient washing with acetonitrile water solution containing sodium acetate after the sample loading is finished.
Further preferably, the sample loading amount of the step (2) is 8-15g/L resin, and the sample loading flow rate is 1-2 times of the volume of the column bed per hour
Further preferably, the gradient wash procedure is as follows: washing with 15-20% acetonitrile water solution (containing 3-4 g/L of sodium acetate and with the pH value of 4.0-4.5), then washing with 15-20% acetonitrile water solution (containing 3-4 g/L of sodium acetate and with the pH value of 5.0-5.5), and finally eluting with 25-30% acetonitrile water solution (containing 3-4 g/L of sodium acetate and with the pH value of 5.5-6.0); and collecting elution effluent containing daptomycin.
Furthermore, the flow rate of the gradient flushing process is 0.5-2 times of the volume of the column bed per hour.
Preferably, the ion exchange resin in the step (3) is NQ-M resin; the ratio of the diameter to the height of the resin column is 1: 3-4.
Preferably, the ion exchange resin chromatography process of step (3) comprises resin column equilibration, loading ion exchange resin column, gradient washing process; the gradient flushing process refers to gradient flushing with sodium chloride solution containing sodium acetate after the sample loading is finished.
Further preferably, the loading amount of the resin in the step (3) is 6-12g/L, and the pH of the loading solution is 6.0-6.5; the sample loading flow rate is 1-3 times of the volume of the column bed per hour.
Further preferably, the step (3) gradient washing process 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.
Furthermore, the flow rate of the gradient flushing process in the step (3) is 1-3 times of the volume of the column bed per hour.
Preferably, the XR30SP resin is used to desalt and remove endotoxin from the collected liquid obtained in step (3) in step (4); the ratio of the diameter to the height of the resin column is 1: 1-2.
Further preferably, the step (4) comprises adjusting the pH of the collected liquid obtained in the step (3) to 2.6-3.6, loading the collected liquid into a hydrophobic resin column, washing the hydrophobic resin column with 10-20% ethanol aqueous solution after loading is finished, and then eluting the hydrophobic resin column with 75-85% ethanol aqueous solution. The sampling flow rate is 2-5 times of the volume of the column bed per hour, and the gradient flushing flow rate is 3-6 times of the volume of the column bed per hour.
Preferably, the nanofiltration concentration process in the step (5) adopts a polyethersulfone nanofiltration membrane, and the molecular weight cutoff is 200-1000 daltons.
Further preferably, in the step (5), the collected solution in the step (4) is diluted by 2-4 times by using water for injection, the concentrated solution is washed by using water for injection in the nanofiltration process with the pH value being adjusted to 2.6-4.6 until the ethanol content in the concentrated solution is lower than 0.05%, and nanofiltration concentration is continued until the daptomycin content in the concentrated solution is higher than 90000mg/L, and the nanofiltration is stopped.
Preferably, the freeze drying process in the step (6) is to freeze for 2-8 hours at-60 ℃ to-45 ℃ and under the pressure of less than 20 Pa; then freeze-drying for 50-80 h at-60-20 ℃ and under the pressure of 20-60 pa; and finally, freeze-drying for 2-8 h at the temperature of 20-30 ℃ and under the pressure of less than 10 pa.
The daptomycin purification method provided by the invention is simple to operate, short in steps, short in production period, high in product yield, high in daptomycin purity and beneficial to large-scale production.
Detailed Description
The present invention is clearly illustrated by the following specific examples, which are to be understood as being illustrative only and not limiting to the scope of the invention.
The fermentation broth sources used in the following examples are referred to in the art for making daptomycin fermentation broths. Other materials, starting materials, reagents, and reagents may be commercially available without specific reference.
Example 1
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the content of daptomycin is 1.6g/L), 3L of ethanol is added, the filtration is carried out by a ceramic membrane with the diameter 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, the filtration is stopped when the content of daptomycin in the ceramic membrane permeation solution is detected by HPLC (high performance liquid chromatography), and 70.1L of permeation solution containing 43.6g of daptomycin is collected.
2) Hydrophobic resin chromatography
The permeate obtained by filtering the ceramic membrane is loaded on an XR3SP resin column (the resin dosage is 5L, the diameter-height ratio is 1:3), the loading flow rate is 1 time of the volume of a column bed per hour, after the loading is finished, 20% acetonitrile solution (pH4.5, containing 3.5g/L of sodium acetate) is used for washing 3BV, 20% acetonitrile solution (pH5.5, containing 3.5g/L of sodium acetate) is used for washing 8BV, 25% acetonitrile solution (pH6.0, containing 3.5g/L of sodium acetate) is used for elution, the collection is started after the peak is absorbed by online ultraviolet monitoring, 14.6L of eluent is collected altogether, the HPLC detection purity is 91.8%, and the content of daptomycin is 34.8 g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH value to 6.5, loading the collected liquid on an NM-Q resin column (the resin dosage is 4L, and the height ratio of the resin column diameter is 1:3), and enabling the flow rate to be 2 times of the volume of the column bed per hour. Washing impurities by using 26BV of 1.7g/L sodium chloride solution (containing 1.6g/L sodium acetate and adjusting the pH value by using acetic acid to be 5.5), then eluting by using 7g/L sodium chloride solution (containing 1.6g/L sodium acetate and adjusting the pH value by using acetic acid) at the flow rate of 2 times of the volume of a column bed per hour, starting to collect the mixture after the peak is absorbed by online ultraviolet monitoring, collecting 8.9L eluent in total, and detecting the purity by using HPLC (high performance liquid chromatography) to be 99.8 percent and containing 27.1g daptomycin.
4) Hydrophobic resin chromatography desalting and endotoxin removing
Adjusting the pH of the collected liquid obtained in the step 3) to 3.2, and loading the collected liquid into an XR30SP resin column (the dosage of the resin is 0.6L, and the diameter-height ratio is 1: 2) the flow rate is 3 BV/h. Washing the resin column with 4BV 10% ethanol solution, eluting with 75% ethanol solution, collecting after the peak is absorbed by on-line ultraviolet monitoring, stopping collecting when the effluent liquid becomes colorless, collecting 650ml of eluent, detecting with HPLC with purity of 99.8%, containing 26.0g daptomycin, and detecting endotoxin content with limulus reagent method of less than 0.06 EU/mg.
5) Nanofiltration concentration
Adding water for injection into the collected liquid obtained in the step 4) to dilute the collected liquid to 1.5L, adjusting the pH value to 3.8 by hydrochloric acid, performing nanofiltration by using a polyethersulfone nanofiltration membrane with the molecular weight cutoff of 200 daltons, adding water for injection in the nanofiltration process to wash the nanofiltration concentrated liquid until the ethanol content of the nanofiltration penetrating liquid is lower than 0.05 percent, continuing the nanofiltration until the daptomycin content in the nanofiltration concentrated liquid is higher than 90000mg/L and collecting 280ml of the nanofiltration concentrated liquid.
6) Freeze drying
Freeze-drying the nanofiltration concentrated solution obtained in the step 5) according to the following conditions: freezing for 2-8 hours at-60 to-45 ℃ under the condition that the pressure is lower than 20 Pa; then freeze-drying for 50-80 hours at the temperature of-60-20 ℃ and under the pressure of 20-60 Pa; and finally, freeze-drying for 2-8 hours at the temperature of 20-30 ℃ and under the pressure of less than 10Pa to obtain 23.9g of daptomycin freeze-dried powder with the HPLC purity of 99.7%.
Example 2
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the content of daptomycin is 1.6g/L), 3.5L of ethanol is added, the mixture is filtered by a 50nm ceramic membrane at room temperature, when the volume of a concentrated solution is less than 10L of the volume of the fermentation liquor, purified water is added to the original volume, when the content of daptomycin in a ceramic membrane permeation solution is detected by HPLC (high performance liquid chromatography), the filtration is stopped, 79.3L of permeation solution is collected, and 43.8g of daptomycin is contained.
2) Hydrophobic resin chromatography
The method comprises the steps of loading a permeation solution obtained by filtering a ceramic membrane onto an XR3SP resin column (the resin consumption is 6L, the diameter-height ratio is 1:4), enabling the loading flow rate to be 2 times of the volume of a column bed per hour, washing 3BV by using a 15% acetonitrile solution (pH4.0 and containing 4.0g/L of sodium acetate), washing 8BV by using a 15% acetonitrile solution (pH5.0 and containing 4.0g/L of sodium acetate), eluting by using a 30% acetonitrile solution (pH5.5 and containing 4.0g/L of sodium acetate) sequentially after loading, starting collection after an on-line ultraviolet monitoring absorption peak, collecting 15.3L of eluent altogether, and detecting the purity by HPLC (high performance liquid chromatography) to be 90.6% and containing 33.2g of daptomycin.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, and loading the diluted liquid on an NM-Q resin column (the dosage of the resin is 5.5L, the height ratio of the resin column diameter is 1:4) at the flow rate of 1 time of the volume of the column bed per hour. The impurities are washed by 30BV of 1.2g/L sodium chloride solution (containing 2g/L sodium acetate and acetic acid for adjusting pH to 5.0), then eluted by 4g/L sodium chloride solution (containing 2g/L sodium acetate and pH to 5.0) with the flow rate of 3 times of the volume of a column bed per hour, the collection is started after the peak is absorbed by online ultraviolet monitoring, 9.7L of eluent is collected together, the purity is 99.6 percent by HPLC detection, and the content of daptomycin is 24.9 g.
4) Hydrophobic resin chromatography desalting and endotoxin removing
Adjusting the pH of the collected liquid obtained in the step 3) to be 2.6, and loading the collected liquid to an XR30SP resin column (the dosage of the resin is 450mL, and the diameter-height ratio is 1: 2) the flow rate is 2 BV/h. The resin column is washed by 4BV of 20% ethanol solution, then eluted by 85% ethanol solution, collection is started after peak absorption is carried out on-line ultraviolet monitoring, collection is stopped when effluent is colorless, 500mL of eluent is collected totally, the purity of the eluent is 99.7% by HPLC (high performance liquid chromatography), the content of daptomycin is 23.8g, and the endotoxin content is less than 0.06EU/mg by a limulus reagent method.
5) Nanofiltration concentration
Adding water for injection into the collected liquid obtained in the step 4) to dilute the collected liquid to 2.0L, adjusting the pH value to 4.6 by hydrochloric acid, performing nanofiltration by using a polyethersulfone nanofiltration membrane with the molecular weight cutoff of 1000 daltons, adding water for injection in the nanofiltration process to wash the nanofiltration concentrated liquid until the ethanol content of the nanofiltration penetrating liquid is lower than 0.05 percent, continuing the nanofiltration until the daptomycin content in the nanofiltration concentrated liquid is higher than 90000mg/L and collecting 250ml of the nanofiltration concentrated liquid.
6) Freeze drying
Freeze-drying the nanofiltration concentrated solution obtained in the step 5) according to the following conditions: freezing for 2-8 hours at-60 to-45 ℃ under the condition that the pressure is lower than 20 Pa; then freeze-drying for 50-80 hours at the temperature of-60-20 ℃ and under the pressure of 20-60 Pa; and finally, freeze-drying for 2-8 hours at the temperature of 20-30 ℃ and under the pressure of less than 10Pa to obtain 21.5g of daptomycin freeze-dried powder with the HPLC purity of 99.5%.
Example 3
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the content of daptomycin is 1.6g/L), 2.5L of ethanol is added, the filtration is carried out 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, the filtration is stopped when the content of daptomycin in the ceramic membrane permeation solution is detected by HPLC (high performance liquid chromatography), 84L of permeation solution is collected, and 43.7g of daptomycin is contained.
2) Hydrophobic resin chromatography
The permeate obtained by filtering the ceramic membrane is loaded on an XR30SP resin column (the resin dosage is 3.5L, the diameter-height ratio is 1:3), the loading flow rate is 1 time of the volume of a column bed per hour, after the loading is finished, 15% acetonitrile solution (pH4.5, containing 3.0g/L of sodium acetate) is used for washing 4BV, 15% acetonitrile solution (pH5.5, containing 3.0g/L of sodium acetate) is used for washing 9BV, 30% acetonitrile solution (pH6.0, containing 3.0g/L of sodium acetate) is used for elution, the collection is started after the peak is absorbed by online ultraviolet monitoring, 15.2L of eluent is collected altogether, the HPLC detection purity is 90.9%, and the daptomycin content is 32.5 g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH value to 6.5, loading the collected liquid on an NM-Q resin column (the resin dosage is 3L, and the height ratio of the resin column diameter is 1:3), and enabling the flow rate to be 3 times of the volume of the column bed per hour. The impurities are washed by 20BV of 2.0g/L sodium chloride solution (containing 1.5g/L sodium acetate and acetic acid for adjusting pH to 5.5), then eluted by 8g/L sodium chloride solution (containing 1.5g/L sodium acetate and pH to 5.5), the flow rate is 1 time of the volume of a column bed per hour, the collection is started after the peak is absorbed by online ultraviolet monitoring, 7.2L of eluent is collected, the purity is 99.5 percent by HPLC, and the daptomycin content is 24.3 g.
4) Hydrophobic resin chromatography desalting and endotoxin removing
Adjusting the pH of the collected liquid obtained in the step 3) to be 3.6, and loading the collected liquid to an XR30SP resin column (the dosage of the resin is 500mL, and the diameter-height ratio is 1: 2) the flow rate is 5 BV/h. The resin column is washed by 4BV 10% ethanol solution, then eluted by 75% ethanol solution, the washing and elution flow rate is 6BV/h, after the peak is absorbed by online ultraviolet monitoring, the collection is started, when the effluent liquid 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 0.06 EU/mg.
5) Nanofiltration concentration
Adding water for injection into the eluent collected in the step 4) to dilute the eluent to 1.5L, adjusting the pH value to 4.6 by hydrochloric acid, performing nanofiltration by using a polyethersulfone nanofiltration membrane with the molecular weight cutoff of 500 daltons, adding water for injection in the nanofiltration process to wash the nanofiltration concentrated solution until the ethanol content of the nanofiltration permeate is lower than 0.05 percent, continuing the nanofiltration until the daptomycin content in the nanofiltration concentrated solution is higher than 90000mg/L, and collecting 240ml of the nanofiltration concentrated solution.
6) Freeze drying
Freeze-drying the nanofiltration concentrated solution obtained in the step 5) according to the following conditions: freezing for 2-8 hours at-60 to-45 ℃ under the condition that the pressure is lower than 20 Pa; then freeze-drying for 50-80 hours at the temperature of-60-20 ℃ and under the pressure of 20-60 Pa; and finally, freeze-drying for 2-8 hours at the temperature of 20-30 ℃ and under the pressure of less than 10Pa to obtain 21.4g of daptomycin freeze-dried powder with the HPLC purity of 99.5%.
Example 4
1) Ceramic membrane filtration
Adding 30L of daptomycin fermentation liquor (the content of daptomycin is 1.6g/L), adding 3L of ethanol, filtering by using a 0.2-micron ceramic membrane at room temperature, adding purified water to the original volume when the volume of a concentrated solution is less than 10L of the volume of the fermentation liquor, stopping filtering when the content of daptomycin in a ceramic membrane permeation solution is detected by HPLC (high performance liquid chromatography), and collecting 71.5L of permeation solution containing 43.2g of daptomycin.
2) Hydrophobic resin chromatography
The permeate obtained by filtering the ceramic membrane is loaded on an XR30SP resin column (the resin dosage is 5L, the diameter-height ratio is 1:3), the loading flow rate is 1 time of the volume of a column bed per hour, after the loading is finished, 20% acetonitrile solution (pH4.5, containing 3.5g/L of sodium acetate) is used for washing 3BV, 20% acetonitrile solution (pH5.5, containing 3.5g/L of sodium acetate) is used for washing 8BV, 25% acetonitrile solution (pH6.0, containing 3.5g/L of sodium acetate) is used for elution, the collection is started after the peak is absorbed by online ultraviolet monitoring, 14.9L of eluent is collected altogether, the HPLC detection purity is 91.3%, and the content of daptomycin is 34.0 g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH value to 6.5, loading the collected liquid on an NM-Q resin column (the resin dosage is 4L, and the height ratio of the resin column diameter is 1:3), and enabling the flow rate to be 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, acetic acid to adjust pH to 5.5), eluting with 7g/L sodium chloride solution (containing 1.64g/L sodium acetate, pH to 5.5) at flow rate 2 times of column bed volume/h, collecting eluate 8.6L after on-line ultraviolet monitoring absorption peak, and detecting by HPLC with purity 99.6% and daptomycin content 26.5 g.
4) Hydrophobic resin chromatography desalting and endotoxin removing
Adjusting the pH of the collected liquid obtained in the step 3) to be 3.2, and loading the collected liquid to an XR30SP resin column (the dosage of the resin is 500mL, and the diameter-height ratio is 1: 1) the flow rate is 3 BV/h. Washing the resin column with 4BV 10% ethanol solution, eluting with 75% ethanol solution, collecting after peak absorption by on-line ultraviolet monitoring, stopping collecting when the effluent becomes colorless, collecting 500mL of eluate, detecting purity by HPLC (high performance liquid chromatography) with purity of 99.6%, containing 25.2g of daptomycin, and detecting endotoxin content less than 0.06EU/mg by limulus reagent method.
5) Nanofiltration concentration
Diluting the collected liquid obtained in the step 4) with injection water to 1.5L, adjusting pH to 3.8 with hydrochloric acid, performing nanofiltration with a polyethersulfone nanofiltration membrane with a molecular weight cutoff of 200 daltons, washing the nanofiltration concentrated liquid with injection water flow in the nanofiltration process until the ethanol content of the nanofiltration permeate is lower than 0.05%, continuing nanofiltration until the daptomycin content in the nanofiltration concentrated liquid is higher than 90000mg/L, and collecting 265ml of the nanofiltration concentrated liquid
6) Freeze drying
Freeze-drying the nanofiltration concentrated solution obtained in the step 5) according to the following conditions: freezing for 2-8 hours at-60 to-45 ℃ under the condition that the pressure is lower than 20 Pa; then freeze-drying for 50-80 hours at the temperature of-60-20 ℃ and under the pressure of 20-60 Pa; and finally, freeze-drying for 2-8 hours at the temperature of 20-30 ℃ and under the pressure of less than 10Pa to obtain 23.1g of daptomycin freeze-dried powder with the HPLC purity of 99.5%.
Example 5
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the content of daptomycin is 1.6g/L), 3L of ethanol is added, the filtration is carried out by a ceramic membrane with the diameter 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, the filtration is stopped when the content of daptomycin in the ceramic membrane permeation solution is detected by HPLC (high performance liquid chromatography), and 70.2L of permeation solution containing 43.3g of daptomycin is collected.
2) Hydrophobic resin chromatography
And (2) filtering a ceramic membrane to obtain a permeate, loading the permeate to a No. three resin column (the resin dosage is 5L, the diameter-height ratio is 1:3), wherein the loading flow rate is 1 time of the volume of a column bed per hour, after the loading is finished, sequentially washing 3BV with 20% acetonitrile solution (pH4.5, containing 3.5g/L of sodium acetate), washing 8BV with 20% acetonitrile solution (pH5.5, containing 3.5g/L of sodium acetate), then eluting with 25% acetonitrile solution (pH6.0, containing 3.5g/L of sodium acetate), collecting after an on-line ultraviolet monitoring absorption peak, collecting 15.2L of eluent in total, and detecting the purity by HPLC (high performance liquid chromatography) of 89.1 percent and the content of daptomycin of 31.6 g.
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 15L of purified water, adjusting the pH value to 6.5, loading the collected liquid on an FPDA-13 resin column (the resin dosage is 4L, and the height ratio of the resin column diameter is 1:3), and enabling the flow rate to be 2 times of the volume of the column bed per hour. Washing impurities by using 26BV of 1.75g/L sodium chloride solution (containing 1.64g/L sodium acetate and adjusting the pH value by using acetic acid to be 5.5), then eluting by using 7g/L sodium chloride solution (containing 1.64g/L sodium acetate and adjusting the pH value by using acetic acid) at the flow rate of 2 times of the volume of a column bed per hour, starting to collect the mixture after the peak is absorbed by online ultraviolet monitoring, collecting 8.1L eluent, and detecting the purity by using HPLC (high performance liquid chromatography) to be 99.3 percent and containing 23.7g daptomycin.
4) Hydrophobic resin chromatography desalting and endotoxin removing
Adjusting the pH of the collected solution obtained in the step 3) to be 3.2, and loading the solution to an HP20SS resin column (the using amount of the resin is 500mL, and the diameter-height ratio is 1: 1) the flow rate is 3 BV/h. The resin column is washed by 4BV 10% ethanol solution, then eluted by 75% ethanol solution, collection is started after peak absorption is carried out by online ultraviolet monitoring, collection is stopped when effluent is colorless, 505mL of eluent is collected totally, the purity is 99.2% by HPLC (high performance liquid chromatography), the daptomycin content is 21.7g, and the endotoxin content is less than 0.3EU/mg by limulus reagent detection.
5) Nanofiltration concentration
Adding water for injection into the collected liquid obtained in the step 4) to dilute the collected liquid to 1.5L, adjusting the pH value to 3.8 by hydrochloric acid, performing nanofiltration by using a polyethersulfone nanofiltration membrane with the molecular weight cutoff of 200 daltons, adding water for injection in the nanofiltration process to wash the nanofiltration concentrated liquid until the ethanol content of the nanofiltration penetrating liquid is lower than 0.05 percent, continuing the nanofiltration until the daptomycin content in the nanofiltration concentrated liquid is higher than 90000mg/L, and collecting 230ml of the nanofiltration concentrated liquid.
6) Freeze drying
Freeze-drying the nanofiltration concentrated solution obtained in the step 5) according to the following conditions: freezing for 2-8 hours at-60 to-45 ℃ under the condition that the pressure is lower than 20 Pa; then freeze-drying for 50-80 hours at the temperature of-60-20 ℃ and under the pressure of 20-60 Pa; and finally, freeze-drying for 2-8 hours at the temperature of 20-30 ℃ and under the pressure of less than 10Pa to obtain 19.5g of daptomycin freeze-dried powder with the HPLC purity of 99.3%.
Example 6
1) Ceramic membrane filtration
30L of daptomycin fermentation liquor (the content of daptomycin is 1.6g/L), 3L of ethanol is added, the filtration is carried out by a ceramic membrane with the diameter 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, the filtration is stopped when the content of daptomycin in the ceramic membrane permeation solution is detected by HPLC (high performance liquid chromatography), and 70.1L of permeation solution containing 43.4g of daptomycin is collected.
2) Hydrophobic resin chromatography
The permeate obtained by filtering the ceramic membrane is loaded on an XR30SP resin column (the resin dosage is 5L, the diameter-height ratio is 1:3), the loading flow rate is 1 time of the volume of a column bed per hour, after the loading is finished, the column is washed by 33% methanol (pH5.5, containing 2.5g/L of sodium acetate) for 10BV, then eluted by 50% methanol (pH6.5, containing 2.5g/L of sodium acetate), the collection is started after the absorption peak is monitored by online ultraviolet, 10.4L of eluent is collected altogether, and the purity is 89.4 percent and the daptomycin content is 34.6g by HPLC (high performance liquid chromatography).
3) Ion exchange resin chromatography
Diluting the collected liquid obtained in the step 2) with 20L of purified water, adjusting the pH value to 6.5, loading the collected liquid on an NM-Q resin column (the resin dosage is 4L, and the height ratio of the resin column diameter is 1:3), and enabling the flow rate to be 2 times of the volume of the column bed per hour. Eluting with 5.8g/L sodium chloride solution (containing 2.5g/L sodium acetate) at flow rate 2 times the volume of the column bed per hour, collecting eluate 7.2L after the peak is absorbed by on-line ultraviolet monitoring, and detecting by HPLC with purity of 99.0% and daptomycin content of 23.3 g.
4) Hydrophobic resin chromatography desalting and endotoxin removing
Adjusting the pH value of the collected liquid obtained in the step 3) to 3.2, loading the collected liquid into an HZ016 resin column (the resin dosage is 500mL, the diameter-height ratio is 1: 1) the flow rate is 3 BV/h. Washing the resin column with 4BV 10% ethanol solution, eluting with 75% ethanol solution, collecting after peak absorption by on-line ultraviolet monitoring, stopping collecting when the effluent becomes colorless, collecting 600L eluate, detecting purity by HPLC (high performance liquid chromatography) with purity of 99.1%, containing 22.1g daptomycin, and detecting endotoxin content less than 0.3EU/mg by limulus reagent method.
5) Nanofiltration concentration
Adding water for injection into the collected liquid obtained in the step 4) to dilute the collected liquid to 1.5L, adjusting the pH value to 3.8 by hydrochloric acid, performing nanofiltration by using a polyethersulfone nanofiltration membrane with the molecular weight cutoff of 200 daltons, adding water for injection in the nanofiltration process to wash the nanofiltration concentrated liquid until the ethanol content of the nanofiltration penetrating liquid is lower than 0.05 percent, continuing the nanofiltration until the daptomycin content in the nanofiltration concentrated liquid is higher than 90000mg/L and collecting 250ml of the nanofiltration concentrated liquid.
6) Freeze drying
Freeze-drying the nanofiltration concentrated solution obtained in the step 5) according to the following conditions: freezing for 2-8 hours at-60 to-45 ℃ under the condition that the pressure is lower than 20 Pa; then freeze-drying for 50-80 hours at the temperature of-60-20 ℃ and under the pressure of 20-60 Pa; and finally, freeze-drying for 2-8 hours at the temperature of 20-30 ℃ and under the pressure of less than 10Pa to obtain 20.1g of daptomycin freeze-dried powder with the HPLC purity of 99.0%.
Comparative example 1
Cooling 30L of daptomycin fermentation liquor (the content of daptomycin is 1.6g/L) to room temperature, clarifying with 0.2 μm ceramic membrane, washing with purified water continuously during the filtration process, removing bacterial residues, and collecting 89L of clarified liquor;
purifying the clarified solution with a 5L HP20 resin column, eluting with 33% methanol at pH5.5 containing 30mM sodium acetate and 50% methanol at pH6.5 containing 30mM sodium acetate, and collecting 9L daptomycin eluate with a purity of > 92%;
diluting the collected liquid 9L with purified water to 10% methanol, purifying with 5LHP20SS resin, eluting with methanol with 30mM sodium acetate at pH5.5 and 45% methanol with 30mM sodium acetate at pH6.5 to obtain daptomycin eluate 8L with purity of more than 94%;
purifying the 8L of the collected liquid by FPDA-13 ion exchange resin, removing the organic solvent, and eluting with 1M sodium chloride and 30mM sodium acetate to obtain 7L of daptomycin eluted collected liquid with the purity of more than 96%;
adjusting pH of the collected feed liquid to 6.0, concentrating by a nanofiltration membrane system with molecular weight cutoff of 360 daltons, and removing small molecular impurities to obtain 500ml of daptomycin concentrated solution; removing endotoxin from the concentrated solution by an ultrafiltration membrane system with the molecular weight cutoff of 10KD, collecting the filtrate, and detecting the endotoxin content by a limulus reagent method to be less than 0.3 EU/mg; concentrating the filtrate to 60g/L, adjusting pH to 6.0, adding isopropanol for precipitation, and collecting precipitate by filtration;
the collected precipitate was dissolved in 10L of water, pH5.0 was adjusted, and the effluent was collected after desalting with cationic resin HZ 016. The obtained effluent is subjected to nanofiltration concentration, sterilized and filtered by a 0.22 mu m membrane, and then is filled and stored at the temperature of minus 20 ℃ to obtain a daptomycin frozen block of 16.8g and a daptomycin HPLC purity of 98.3%.
Comparative example 2
Adjusting the pH value of daptomycin fermentation liquor (30L, the content of daptomycin is 1.6g/L) to 3.0 by using phosphoric acid, filtering by using a ceramic membrane with the pore diameter of 0.05 mu m to remove macromolecular substances such as water-soluble protein, pigment and the like in the daptomycin fermentation liquor, circularly washing the ceramic membrane by using 1-2 times of volume of phosphoric acid aqueous solution with the pH value of 3.0 of raw material liquid (discarding filtrate), circularly washing the ceramic membrane by using 4-5 times of volume of sodium hydroxide solution with the pH value of 9.0 of the raw material liquid to obtain ceramic membrane filtrate, and clarifying and enabling the obtained filtrate to be reddish yellow.
When the pH value of the filtrate is adjusted to 6.0 by dilute acetic acid, the filtrate passes through a small segment of D101 resin column (the ratio of diameter to height is 1:10) once, the series connection is disconnected, each small segment of the column is respectively washed by water until the effluent liquid of each segment is basically colorless, and then 3 small segments of the D101 resin column are sequentially connected in series and then connected in series with another D101 resin column (the ratio of diameter to height is 1: 10); and eluted with 0.06N sodium acetate, 30% ethanol solution, pH 6.5.
The eluate is subjected to D301 resin column (diameter/height ratio of 1:10) at pH5.5 with diluted acetic acid, washed with water, and then eluted with 300mM sodium chloride solution at pH 5.5-6.0. And (3) nanofiltration and concentration of D301 resin column eluate by a nanofiltration membrane with the molecular weight cutoff of 100-200, wherein the concentration of daptomycin after concentration is 20% (w/w). And (4) carrying out vacuum freeze-drying on the concentrated solution to obtain 18.4g of daptomycin freeze-dried powder with the HPLC purity of 97.5%.
Claims (10)
1. A daptomycin purification method is characterized by comprising the following steps:
(1) filtering the daptomycin fermentation liquor by using a ceramic membrane;
(2) carrying out hydrophobic resin chromatography;
(3) ion exchange resin chromatography;
(4) separating out salt and endotoxin by using a hydrophobic resin layer;
(5) nano-filtering and concentrating;
(6) and (5) freeze drying.
2. The method of claim 1, wherein the hydrophobic resin of step (2) is an XR3SP resin or an XR30SP resin.
3. The method according to claim 1, wherein the hydrophobic resin chromatography step of step (2) comprises a resin column equilibration, loading, gradient washing process; the gradient washing process is to use acetonitrile water solution containing sodium acetate to carry out gradient washing after the loading is finished.
4. The method of claim 3, wherein the gradient wash procedure is as follows: washing with 15-20% acetonitrile water solution with pH of 4.0-4.5 and 3-4 g/L sodium acetate, washing with 15-20% acetonitrile water solution with pH of 5.0-5.5 and 3-4 g/L sodium acetate, and eluting with 25-30% acetonitrile water solution with pH of 5.5-6.0 and 3-4 g/L sodium acetate; and collecting elution effluent containing daptomycin.
5. The method according to claim 1, wherein the ion exchange resin of step (3) is NQ-M resin.
6. The method according to claim 1, wherein the ion exchange resin chromatography process of step (3) comprises a resin column equilibration, loading, gradient washing process; the gradient washing process is to use sodium chloride solution containing sodium acetate to carry out gradient washing after the sample loading is finished.
7. The method according to claim 6, wherein the gradient washing process comprises washing with 1.2-3 g/L sodium chloride solution containing 1.5-2.0 g/L sodium acetate at pH 5.0-5.5, and then eluting with 4-8 g/L sodium chloride solution containing 1.5-2.0 g/L sodium acetate at pH 5.0-5.5; and collecting elution effluent containing daptomycin.
8. The method of claim 1, wherein the hydrophobic resin of step (4) is an XR30SP resin.
9. The method as claimed in claim 1, wherein the step (4) comprises adjusting the pH of the collected solution obtained in the step (3) to 2.6-3.6, loading the hydrophobic resin column, washing the hydrophobic resin column with 10-20% ethanol solution after loading, eluting with 75-85% ethanol solution, and collecting the elution effluent containing daptomycin.
10. The method as claimed in claim 1, wherein step (5) is carried out by nanofiltration concentration with polyethersulfone nanofiltration membrane, wherein the molecular weight cut-off of the nanofiltration membrane is 200-1000 daltons.
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| CN113717253A (en) * | 2021-09-15 | 2021-11-30 | 丽珠集团福州福兴医药有限公司 | Purification method of daptomycin |
| CN113866330A (en) * | 2021-10-26 | 2021-12-31 | 丽珠集团福州福兴医药有限公司 | Separation and purification method and application of dehydrated 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 |
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| CN113717253A (en) * | 2021-09-15 | 2021-11-30 | 丽珠集团福州福兴医药有限公司 | Purification method of daptomycin |
| CN113866330A (en) * | 2021-10-26 | 2021-12-31 | 丽珠集团福州福兴医药有限公司 | Separation and purification method and application of dehydrated daptomycin |
| CN113866330B (en) * | 2021-10-26 | 2023-08-22 | 丽珠集团福州福兴医药有限公司 | Separation and purification method and application of dehydrated 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 |
| 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 |
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