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WO2019168421A1 - Procédé d'isolement d'un composant de sulfate de polymyxine e pur - Google Patents

Procédé d'isolement d'un composant de sulfate de polymyxine e pur Download PDF

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Publication number
WO2019168421A1
WO2019168421A1 PCT/PL2019/050012 PL2019050012W WO2019168421A1 WO 2019168421 A1 WO2019168421 A1 WO 2019168421A1 PL 2019050012 W PL2019050012 W PL 2019050012W WO 2019168421 A1 WO2019168421 A1 WO 2019168421A1
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WIPO (PCT)
Prior art keywords
sulfate
polymyxin
vol
colistin
column
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Ceased
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PCT/PL2019/050012
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English (en)
Inventor
Bartłomiej FEDORCZYK
Aleksandra MISICKA-KĘSIK
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Uniwersytet Warszawski
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Uniwersytet Warszawski
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Publication of WO2019168421A1 publication Critical patent/WO2019168421A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related peptides

Definitions

  • the present invention relates to a method for isolation of pure polymyxin E sulfate component, especially selected from: polymyxin E1 sulfate or polymyxin E2 sulfate.
  • Polymyxin E (also known as colistin) has been known for over 60 years as an antibiotic produced biosynthetically by microorganism Bacillus polymyxa var. colistinus. It is a mixture of peptides possessing basic character. The components of the mixture are polymyxins: E1 , E2, E3, E4, E1-I, E2-I, E6, E1-Nva, 2,3-dehydro E1 and E1-7MOA, shown in Formula 1 , with the dominant components being polymyxin E1 and polymyxin E2.
  • Purified colistin usually contains about 60% polymyxin E1. It is a fact that mixtures from different production sources may differ in their composition, which may also significantly differ from the accepted norms regarding the percentage of individual components, mainly polymyxins E2 and E1 , but also other components with a smaller share.
  • Colistin sulfate obtained by treating purified colistin or semisynthetic sodium colistimethate with sulfuric acid, is used in the preparation of pharmaceutical formulations.
  • nephrotoxicity and neurotoxicity When used intravenously, the main types of toxicity seen with colistin are nephrotoxicity and neurotoxicity. It can be assumed that some of them are caused by components of polymyxin E, which are not essential for obtaining its antimicrobial activity. It should also be expected that the purification of the most important active ingredients of polymyxin E would allow to establish the exact composition of the mixture used in medicine obtained by mixing them in specific and optimal proportions.
  • EP2470555 discloses a method for purification of colistin by reversed phase chromatography, wherein the colistin base is separated using an eluent system consisting of an aqueous ethanol solution with a small amount of acetic acid.
  • An object of the invention is to provide a method for isolation of polymyxin E1 and polymyxin E2 sulfates that could be used to develop a modern antibiotic formulation with a strictly defined composition and antimicrobial activity, allowing its precise dosing and at least partial avoidance of complications related to impurities and incorrect dosing.
  • a particular object of the invention is to propose a chromatographic method for separation of crude colistin mixture, which could be used on an industrial scale. It is particularly desirable to obtain the maximum separation of E1 and E2 fractions and to avoid the use of toxic methanol. It is a particular object of the invention to provide a process for obtaining pure polymyxin E1 sulfate and pure polymyxin E2 sulfate from a mixture comprising these substances, in particular from the formulation of polymyxin E sulfate.
  • the present invention relates to a method for preparation of pure polymyxin E sulfate component, especially selected from: polymyxin E1 sulfate or polymyxin E2 sulfate, by reversed phase chromatography, characterized in that the 3 ⁇ 4queous solution of the mixture comprising polymyxin E1 sulfate and polymyxin E2 sulfate and from 0.01 to 5 vol%, preferably 0.1 % formic acid is applied to a chromatographic column filled with C12 resin and then eluted with an aqueous solution of acetonitrile containing from 0.01 to 5 vol%, preferably 0.1 % formic acid, wherein the effluent fraction containing the selected polymyxin E sulfate component is identified and collected, preferably polymyxin E1 sulfate or polymyxin E2 sulfate.
  • elution is carried out with a gradient of acetonitrile concentration linearly increasing from 1 to 61 vol%, wherein elution is carried out for a period of 5 to 100 minutes.
  • elution is carried out with a constant concentration of acetonitrile selected from the range of 5 to 50 vol%.
  • fraction containing selected component is identified by analyzing the effluent by mass spectrometry or by performing an absorbance measurement at 215 nm.
  • the mixture comprising polymyxin E1 sulfate and polymyxin E2 sulfate is colistin sulfate.
  • the chromatographic separation is carried out by HPLC, using a flow rate ranging from 0.5 ml/min to 1.5 ml/min on a column with a diameter of 4.6 mm and a length of 250 mm.
  • Increasing the diameter of the chromatographic column allows increasing the flow rate. This particularly applies to the embodiment of the invention in which the separation is carried out by HPLC.
  • the flow rate should then be adjusted in proportion to the square of the diameter of the column used.
  • the new flow rate can be calculated from the following formula:
  • Fi is a flow rate for column 1
  • F 2 is a flow rate for column 2
  • d 2 is a diameter of column 2
  • pH of the mobile phase is ⁇ 4, preferably from 2 to 2.5.
  • the selected polymyxin E sulfate component is additionally isolated from the fractions containing it by precipitation at pH from 1 1 to 12, followed by lyophilization, and redissolution in a solution of sulfuric acid at pH from 3 to 5, followed by re-lyophilization.
  • the disclosed method is suitable for efficient separation of individual components of polymyxin E sulfate and can be used in industry.
  • a particularly preferred embodiment of the method of the invention is preparative HPLC.
  • polymyxin E1 sulfate of 78.9% purity is obtained in 97.9% yield
  • polymyxin E2 sulfate of 90.8% purity is obtained in 87.5% yield.
  • Higher purity >90% is possible by repeating chromatographic separation.
  • the method according to the invention can be used to transform the raw material that does not meet the standards into the raw material that suits them.
  • Fig. 1 shows a chromatogram for the initial colistin sulfate obtained in the method according to Ph. Eur. 28.4 Colistin Sulfate. Peaks with RT 9.5 min and with RT 18.0 min correspond to polymyxin E2 (57.2%) and E1 (27.9%).
  • FIG. 2 shows a chromatogram for a certified reference material (EPRS, Y0000277, batch 4.0) obtained in the method according to Ph. Eur. 28.4 Colistin Sulfate. Peaks with RT 9.4 min and with RT 18.0 min correspond to polymyxin E2 (37.1 %) and E1 (46.3%).
  • EPRS certified reference material
  • the polymyxin E separation was carried out using a reversed phase high- performance liquid chromatography technique. Selection of appropriate conditions for the separation of colistin sulfate on a preparative scale was made. For this purpose, C-12 type columns with dimensions of 4.6 x 250 mm and a grain diameter of 4 microns were used. The method was then scaled to a 21.2 x 250 mm preparative column filled with the same bed.
  • the mass spectrometry method was used to detect the effluent from the column, in which the presence of pseudo-molecular ions [M+3H] 3+ identical to the main constituents of colistin sulfate, polimyksyn E2 (m/z 385.92) and E1 (m/z 390.59) as well as total ion current (TIC) was monitored.
  • the fact is that some of the other colistin sulfate components have the same pseudo- molecular ion values, but they differ in both retention time on the column and the area under the signal. Due to the above, they can be distinguished from each other.
  • the methods were tested for the possibility of overloading the column bed by applying very large amounts of colistin sulfate to a single chromatographic process. This is a key aspect in preparative runs because it saves time and resources used for the separation process.
  • Fig. 3 shows the chromatogram for isolated E2 colistin obtained in the method according to Ph. Eur. 28.4. Peak E2 with RT 9.5 min (93.6%), RT 8.8 min (1.8%), RT 10.5 min (E3 - 2.1 %), residual impurities ⁇ 0.6%.
  • Fig. 4 shows the chromatogram for isolated E1 colistin obtained in the method according to Ph. Eur. 28.4. Peak E1 with RT 18.1 min (93.8%), RT 16.6 min (2,3- dehydro E1 - 2.2%), RT 19.6 min (E1-7MOA - 3.3%), residual impurities ⁇ 0.3%.
  • Example 1 Testing of various HPLC conditions on an analytical scale.
  • phase B gradient purity acetonitrile with 0.1 vol% addition of formic acid.
  • the flow rate was set to 1 ml/min, the separation was carried out at room temperature in the range of 22-25 °C.
  • Fig. 5 shows a plot of the total ion current from which the signal measured for pseudomolecular ions was isolated for polymyxin E2 and E1 in the gradient method A with 2.5 pg of colistin sulfate loaded.
  • Fig. 6 shows a plot of the total ion current from which the signal measured for pseudomolecular ions was isolated for polymyxin E2 and E1 in the gradient method B with 2.5 pg of colistin sulfate loaded.
  • Fig. 7 shows a plot of the total ion current from which the signal measured for pseudomolecular ions was isolated for polymyxin E2 and E1 in the gradient method C with 2.5 pg of colistin sulfate loaded.
  • Fig. 8 shows a plot of total ion current from which the signal for pseudomolecular ions was measured for polymyxin E2 and E1 in the gradient method C with 2.5 mg of colistin sulfate loaded.
  • isocratic elution which is carried out using an aqueous solution of acetonitrile at a concentration ranging between 1 % and 61 % with the addition of 0.1 vol% formic acid, depending on the mass of colistin sulfate loaded.
  • An example of a separation method using isocratic elution is method D, in which all the hardware parameters are preserved as in the case of gradient methods A-C. The only exception was the separate preparation of the solution for eluting the compounds from the column, which is prepared by mixing 91 volumes of phase A and 9 volumes of phase B. It is also possible to mix the phases to achieve the preset solvent system using a mixer on the high pressure side in the system. Fig.
  • FIG. 9 shows an exemplary isocratic run for colistin sulfate on the analytical column in method D.
  • the chromatogram presents a plot of the total ion current registered for pseudomolecular ions in the range of 220-2000 m/z in both polarizations (+/-).
  • a column of dimensions 21.2 x 250 mm with the same bed as the analytical column was used to scale-up the colistin sulfate separation method. Isocratic elution was applied.
  • Fig. 10 shows plots of the total ion current for FIPLC-MS analyzes of the collected fractions 1-12 after the preparative run, obtained in method D.
  • Fig. 1 1 shows plots of the total ion current for HPLC-MS analyzes of collected fractions 13-23 after the preparative run, obtained in method D.
  • Example 3 Isolation of selected polymyxin E sulfates from fractions after preparative run.
  • Fig. 12 shows the chromatogram obtained for the E1 sulfate fraction in the method according to Ph. Eur. 28.4 Colistin Sulfate. Composition: ⁇ 2 - RT 9.4 min (5.1 %), E3 - RT 10.4 min (1.7%), 2,3-dehydro E1 - RT 16.5 min (2.1 %), E1 - RT 18.0 min (78.9%), E1-7MOA - RT 19.5 min (10.4%).
  • Fig. 13 shows the chromatogram obtained for the E2 sulfate fraction in the method according to Ph. Eur. 28.4 Colistin Sulfate. Composition: E2 - RT 9.4 min (90.8%), E3 - RT 10.4 min (1.9%).
  • the developed method allows obtaining a raw material in which the content of the desired polymyxin E1 is increased from the initial 27.9% to 78.9%. From 2000 mg of raw material (558 mg of pure E1 ) loaded onto the column, 692.5 mg of E1 sulfate fraction (546.3 mg of pure E1 ) is obtained, hence, the recovery of the substance is quantitative and amounts to 97.9%. In case a higher purity of polymyxin E1 is desired, chromatographic separation should be performed again and the side fractions should be rejected (see Fig. 4).

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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)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé d'obtention d'un composant de sulfate de polymyxine E pur, particulièrement choisi parmi : le sulfate de polymyxine E1 ou le sulfate de polymyxine E2, par chromatographie en phase inverse, notamment CLHP préparatoire, selon laquelle une solution aqueuse d'un mélange comprenant du sulfate de polymyxine E1 et du sulfate de polymyxine E2, en particulier une solution aqueuse de sulfate de colistine est chargée sur une colonne de chromatographie et ensuite éluée avec l'éluant, la fraction d'effluent contenant le composant sélectionné étant collectée. Dans le procédé selon l'invention, il est possible de charger de grandes quantités de la préparation séparée sur la colonne. L'invention étant ainsi utilisée pour réaliser une séparation à l'échelle industrielle.
PCT/PL2019/050012 2018-02-28 2019-02-28 Procédé d'isolement d'un composant de sulfate de polymyxine e pur Ceased WO2019168421A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PLP.424712 2018-02-28
PL424712A PL239311B1 (pl) 2018-02-28 2018-02-28 Sposób izolowania czystego składnika siarczanu polimyksyny E

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011051070A1 (fr) * 2009-10-30 2011-05-05 Xellia Pharmaceuticals Aps Procédé de purification de la colistine et composants à base de colistine purifiée
CN103396475A (zh) * 2013-08-06 2013-11-20 深圳翰宇药业股份有限公司 一种纯固相合成多肽类抗生素Colistin的方法
CN104672309A (zh) * 2013-11-29 2015-06-03 江苏汉邦科技有限公司 一种硫酸粘菌素的纯化方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011051070A1 (fr) * 2009-10-30 2011-05-05 Xellia Pharmaceuticals Aps Procédé de purification de la colistine et composants à base de colistine purifiée
CN103396475A (zh) * 2013-08-06 2013-11-20 深圳翰宇药业股份有限公司 一种纯固相合成多肽类抗生素Colistin的方法
CN104672309A (zh) * 2013-11-29 2015-06-03 江苏汉邦科技有限公司 一种硫酸粘菌素的纯化方法

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PL239311B1 (pl) 2021-11-22

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