WO2019168421A1

WO2019168421A1 - A method for isolation of pure polymyxin e sulfate component

Info

Publication number: WO2019168421A1
Application number: PCT/PL2019/050012
Authority: WO
Inventors: Bartłomiej FEDORCZYK; Aleksandra MISICKA-KĘSIK
Original assignee: Uniwersytet Warszawski
Current assignee: Uniwersytet Warszawski
Priority date: 2018-02-28
Filing date: 2019-02-28
Publication date: 2019-09-06
Anticipated expiration: 2020-08-28
Also published as: PL424712A1; PL239311B1

Abstract

A method for obtaining a pure polymyxin E sulfate component is disclosed, especially selected from: polymyxin E1 sulfate or polymyxine E2 sulfate, by reversed phase chromatography, especially preparative HPLC, in which an aqueous solution of a mixture comprising polymyxin E1 sulfate and polymyxin E2 sulfate, especially an aqueous solution of colistin sulfate is loaded onto chromatographic column and then eluted with the eluent, wherein the effluent fraction containing the selected component is collected. In the method according to the invention, it is possible to load large quantities of the separated preparation onto the column. This allows the invention to be used to carry out an industrial scale separation.

Description

A method for isolation of pure polymyxin E sulfate component

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.

State of the art

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.

In connection with the currently observed increasing drug resistance to the most commonly used antibiotics, such as b-lactams, there is a growing clinical need for formulations containing less often used active substances that may be useful in the clinical control of infections caused by drug-resistant microorganisms. This leads to increased interest in polymyxin E and raises the need to develop modern antibiotic formulations based on its components.

The classic methods for isolation and purification of colistin sulfate are sorption of the antibiotic from the filtrate of the fermentation broth on the carboxylic cationite in the salt or protonated form, followed by elution of the ion-exchange bed, purification of the eluate and isolation of polymyxin E sulfate from the purified solution by lyophilization or precipitation with organic solvents. Polish patent specification PL77448 describes such a method of obtaining pure polymyxin E sulfate.

For historical reasons, in the case of polymyxin E, there are no such stringent requirements as in the case of modern medicines, in particular in the field of toxicology and the content of contaminants. The binding pharmacopoeia specifies only the required ranges of the content of the predominant constituents and the minimum antimicrobial activity authorized for use in the treatment of colistin. As a result, the compositions of various colistin formulations are significantly different, and each of the manufacturers independently determines the recommended doses, including the maximum allowable dose of the offered formulation. Consequently, determining the correct dosage is very troublesome in clinical practice, especially when administered intravenously.

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

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.

Unexpectedly, such an object has been achieved in the present invention.

The essence of the invention 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 ¾queous 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.

Preferably, 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.

Equally preferably, elution is carried out with a constant concentration of acetonitrile selected from the range of 5 to 50 vol%.

Preferably, fraction containing selected component is identified by analyzing the effluent by mass spectrometry or by performing an absorbance measurement at 215 nm.

In a particular embodiment of the invention, the mixture comprising polymyxin E1 sulfate and polymyxin E2 sulfate is colistin sulfate.

In a preferred embodiment of the method according to the invention, 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.

If the column diameter changes, the new flow rate can be calculated from the following formula:

F₂ = FI ^* (d₂)² / (di)² where:

Fi is a flow rate for column 1 F₂ is a flow rate for column 2

di is a diameter of column 1

d₂ is a diameter of column 2

The following table gives an example of particularly favorable flow rates for columns with different diameters.

Preferably, in the method according to the invention, pH of the mobile phase is <4, preferably from 2 to 2.5.

Preferably, 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.

In the embodiments described below, polymyxin E1 sulfate of 78.9% purity is obtained in 97.9% yield, and polymyxin E2 sulfate of 90.8% purity is obtained in 87.5% yield. Higher purity >90% is possible by repeating chromatographic separation.

Thanks to the choice of eluents, chromatographic resin and other HPLC conditions proposed in accordance with the invention, surprisingly, not only the complete separation of the polymyxin E1 sulfate and the polymyxin E2 sulfate fraction, but also their considerable separation was obtained. In the exemplary embodiment (method D), the interval between the maxima of both fractions is about 10 minutes. As a result, in the method according to the invention, it is possible to apply a large amount of the preparation to be separated on the column. This allows the invention to be used to carry out an industrial scale separation.

If crude polymyxin E is used that does not meet the standards set out in the pharmacopoeia, 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.

Detailed description of the invention

In the embodiments described below, the crude colistin sulfate has been used, which does not meet the pharmacopeia requirements and contains polymyxins E1 and E2, which are in this case at an unsuitable level. 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%).

For comparison, 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%).

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.

Gradual and isocratic runs were tested, and their potential for use in polymyxin E separation on a preparative scale was evaluated. The methods are characterized by loading of colistin sulfate in an aqueous solution of formic acid (0.1 vol%) on chromatographic column, followed by elution with a water/acetonitrile system with the addition of 0.1 vol% formic acid with pH of mobile phase of approximately 2-2.5. It turned out that the chromatographic methods developed allow a good separation of polymyxins E2 and E1 , yielding fractions with purity >90%.

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.

It turned out that the Phenomenex Jupiter Proteo 90A, C-12, 4 pm bed has good selectivity for polymyxins E1 and E2 separation. Experiments were carried out to illustrate the effect of the composition of the eluents and the amount of loaded colistin sulfate on the separation of E1 and E2.

Analytical HPLC:

Reagents used:

- ultrapure MiliQ water with a resistance of 18 MW cm,

- gradient grade acetonitrile Lichrosolv Reag. Ph Eur, Merck

- formic acid for the analysis, EMSURE ACS, Reag. Ph Eur, Merck

- colistin sulfate.

The analysis of potential of gradient and isocratic methods for separation on a preparative scale:

Phenomenex 4.6 x 250 mm column with Jupiter Proteo 90A, C-12, 4 pm bed.

Shimadzu chromatograph:

- LC-20AD pumps,

- automatic sample loading system SIL-20AC,

- CTO-20AC column thermostat,

- CBM-20A interface,

- detector: LCMS-IT-TOF mass spectrometer with an ESI source.

Used eluents: - phase A: MiliQ type water with 0.1 vol% addition of formic acid,

- 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.

Test analytical runs were performed using different gradients by loading 10 mI of colistin sulfate solution in phase A at a concentration of 0.25 mg/ml (2.5 pg of colistin sulfate):

- in gradient method A with a linear increase in the proportion of phase B from 1 % to

61 % in 30 minutes. The results are presented in Fig. 5, which 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.

- in the gradient method B with a linear increase in proportion of phase B from 1 % to

31 % in 30 minutes. The results are presented in Fig. 6, which 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.

- in the gradient method C with a linear increase in proportion of phase B from 8% to

17% in 30 minutes. The results are presented in Fig. 7, which 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.

It turned out that a good selectivity can be obtained for the gradient method C that ensures separation of polymyxins E1 and E2, as well as their major trace impurities, e.g. polymyxin E3 and E1-7MOA.

In addition, runs were also performed in which larger amounts of colistin sulfate were loaded. Unexpectedly, loading 2.5 mg of colistin sulfate (1000x more) and 12.5 mg (5000x more) to the analytical column with dimensions 4.6 x 250 mm still allows almost quantitative separation of compounds that are eluted from the column in a very narrow time interval. 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. In addition to gradient methods, it is preferable to use 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. 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 (+/-).

Example 2. Preparative HPLC.

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.

Phenomenex 21.2 x 250 mm column with Jupiter Proteo 90A, C-12, 4 pm bed.

Shimadzu chromatograph:

- LC-20AP pumps,

- loop for manual loading with volume of 10 ml,

- CBM-20A interface,

- SPD-20A detector set to 215 nm.

Used eluents:

- A solution of acetonitrile in water with a concentration ranging from 1 % to 61 %. Prepared by mixing in a suitable ratio MiliQ water with 0.1 vol% addition of formic acid and gradient grade acetonitrile with 0.1 vol% addition of formic acid.

2 g of colistin sulfate dissolved in MiliQ water, with 0.1 vol% addition of formic acid to a total volume of 10 ml, was loaded on the column. The phases flow rate through the column was scaled-up to 22 ml/min, the separation was carried out at room temperature in the range of 22-25 deg. C, collecting 40 ml effluent fractions starting at RT of approx. 12 min. The starting point for collecting the effluent was determined by the observation of a characteristic increase in the absorbance on the detector. The end point was determined by the decrease and the subsequent equalization of the absorbance on the detector at about 10 mAU. Individual fractions were analyzed by HPLC-MS-IT-TOF using method D.

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.

The fractions with the identified qualitative composition, containing the separated polymyxins E1 and E2, were then combined. Ammonia solution was added dropwise to the solutions to reach pH 1 1-12. Under these conditions, a slow precipitation of the colloidal colistin base is observed. Then, the solution was frozen at -80 °C and lyophilized. The dry fluffy precipitate was suspended in MiliQ water and titrated with a 1 % sulfuric acid solution to pH 3-5, to obtain a clear solution, after which it was frozen again at -80 °C and lyophilized. The dry product was weighed to obtain 1000.9 mg of polymyxin E2 sulfate and 692.5 mg of polymyxin E1 sulfate. A chromatographic analysis was performed according to Ph Eur 28.4 for the Colistin Sulfate substance.

Fig. 12 shows the chromatogram obtained for the E1 sulfate fraction in the method according to Ph. Eur. 28.4 Colistin Sulfate. Composition: Έ2 - 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%).

Conclusions

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).

Claims

1. A method for obtaining a pure polymyxin E sulfate component, especially selected from: polymyxin E1 sulfate or polymyxin E2 sulfate, by reversed phase chromatography, characterized in that the aqueous solution of the mixture comprising polymyxin E1 sulfate and polymyxin E2 sulfate and from 0.01 to 5 vol%, preferably 0.1 vol% formic acid, is loaded onto 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 vol% formic acid, wherein an effluent fraction containing the selected polymyxin E sulfate component is identified and collected, preferably polymyxin E1 sulfate or polymyxin E2 sulfate.

2. The method according to claim 1 , characterized in that the 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.

3. The method according to claim 1 , characterized in that the elution is carried out with a constant concentration of acetonitrile selected from the range of 5 to 50 vol%.

4. The method according to claim 1 , characterized in that the fraction containing the selected ingredient is identified by analyzing the effluent by mass spectrometry or by performing an absorbance measurement at 215 nm.

5. The method according to claim 1 , characterized in that the mixture comprising polymyxin E1 sulfate and polymyxin E2 sulfate is colistin sulfate.

6. The method according to any one of the preceding claims, characterized in that the chromatographic separation is carried out by HPLC, using a flow rate corresponding to flow rate ranging from 0.5 ml/min to 1.5 ml/min on a column with a diameter of 4.6. mm and length 250 mm.

7. A method according to any one of the preceding claims, characterized in that pH of the mobile phase is <4, preferably from 2 to 2.5.

8. A method according to any one of the preceding claims, characterized in that the selected polymyxin E sulfate component is isolated from fractions containing it by precipitation at a pH of 1 1 to 12, followed by lyophilization, and redissolution in a solution of sulfuric acid at a pH of 3 to 5 and re-lyophilization.