Field of the invention

(Int. Cl.: C 07 K 5/12, A 61 K 38/12)

The present invention belongs to the field of medicinal active substances from the group of glycopeptide antibiotics and relates to pure vancomycin hydrochloride substantially free of impurities known in commercially available products and purity of vancomycin hydrochloride ranges from about 97% to about 99%, as well as to the new process for the purification of vancomycin by use of preparative HPLC, that is, the method of displacement chromatography.

Vancomycin hydrochloride is used for the treatment of severe staphylococcal infections, especially those caused by methicillin-resistant staphylococcal strains.

Technical problem

For antibiotics and vancomycin hydrochloride, respectively, because of accompanying impurities and consequently possible side effects, a high purity of the antibiotics is desired for the certain types of use in patients. Therefore, there is a constant need for pure vancomycin hydrochloride substantially free of accompanying impurities and the process, respectively, by which such the product would be prepared.

Prior art

Vancomycin is a tricyclic amphoteric glycopeptide antibiotic used in the therapy in the form of its hydrochloride salt and is also cited in The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals, 13 ^ed (2001), under Monograph no. 9995. It was first disclosed in U.S. Pat. No. 3,067,099. Vancomycin hydrochloride is used for the treatment of staphylococcal infections, especially

infections caused by methicillin-resistant staphylococcal strains. Vancomycin is a fermentation product isolated from a fermentation broth Nocardia orientalis (formerly Streptomyces orientalis such as, for example, Streptomyces orientalis NRRL 2452) which produces a mixture of related co-fermentative factors. "Factor B" (vancomycin B) is identified as the most important antibiotic in the mixture and is available in commercial products. The vancomycin fermentation broth is filtered and the filtrate is added to a column that contains an adsorption resin that decolorizes and desalts vancomycin. The resin is washed and eluted with a solvent of low pH, the eluate is then decolorized with active carbon. The vancomycin eluate is subsequently purified using a crystallization step at low pH of the medium. The crystallized vancomycin is combined with hydrochloric acid and precipitated in an organic solvent such as acetone to form vancomycin hydrochloride.

Vancomycin hydrochloride is used orally or parenterally, and it is in form of a dry substance as a dry off-white powder in sterile vials or small bottles. The dry solid form of vancomycin is obtained by lyophilization of the aqueous solutions of its hydrochloride salt and with water it forms a clear solution having a pH between 2.5 and 4.5.

Literature describes a number of processes for the preparation of vancomycin and hydrochloride salt thereof, respectively, from the fermentation medium disclosed in the above U.S. Pat. No. 3,067,099 and other literature describing precipitation processes with alkali hydroxide, such as sodium hydroxide described in U.S. Pat. No. 5,037,652 or U.S. Pat. No. 5,235,037; processes for the purification with imidazole vancomycin complex, described in U.S. Pat. No. 4,868,285; via the formation of phosphates described in patent EP 145 484, via the formation of complexes with peptides disclosed in U.S. Pat. No. 4,667,024 or by adsorption onto different polymer resins disclosed in U.S. Pat. No. 4,440,753 or U.S. Pat. No. 4,874,843.

U.S. Pat. No. 5,574,135 discloses an improved process for the manufacture of crystalline vancomycin which consists of passing a vancomycin fermentation broth

through two adsorbents successively, producing a purified vancomycin. Purified vancomycin is then crystallized from the solution by adding an alkali base solution to impart a pH between 9.0 and 9.5 to the medium. It is disclosed that the vancomycin obtained is of greater purity than vancomycin produced by other prior art processes. The vancomycin purity is about 90% after two crystallization steps.

U.S. Pat. No. 5,854,390 discloses a new process for the purification of vancomycin by High Pressure Liquid Chromatography, that is, by method of displacement chromatography, whereby chromatographic purity 95.5% area of the vancomycin hydrochloride is essentially improved. The chromatography is performed on a reverse stationary phase with a mobile phase consisting of an organic or inorganic acid or an appropriate buffer, and with different displacing agents, at a defined pH and temperature, and the amount and concentration of vancomycin. According to the disclosed process, the portion of impurities vancomycin hydrochloride contains is for one third lower than in hitherto known commercially available products.

The method of displacement chromatography is known and described in literature, e.g., in the article of G. Subramanian et al., "Displacement Chromatography of Biomolecules", J. of Chromatography, Vol. 439, (1988), pp. 341-351 , and is based on the principle that in a sample the balance between stationary phase and mobile phase is shifted the direction of stationary phase. Single components of the sample displace each other, and the displacing agent of the greater affinity to stationary phase pushes the components of the mixture out of the column.

U.S. Pat. No. 4,885,275 discloses new and stable concentrated aqueous solutions of vancomycin hydrochloride without accompanying gel as well as a new process for lyophilization of vancomycin hydrochloride obtained in the form of dry, freely soluble and flowing powder from the said concentrated solutions.

Description of the invention including examples

An object of the present invention is to solve the problem known in the prior art, that is, to manufacture a new pure vancomycin hydrochloride substantially free of impurities, known in commercially available products, according to the new process for chromatographic purification of vancomycin by which vancomycin hydrochloride of exceptionally high purity will be obtained. The term pure vancomycin hydrochloride "substantially free of impurities" used herein means a purity of about 97% to about 99%, preferably about 98% to about 99%, most preferably about 99%, determined by HPLC analytical method as directed in U.S. Pharmacopoeia, United States Pharmacopoeia, The National Formulary NF, 27 ^th revision, 22 (2004), Monograph: "Vancomycin Hydrochloride". The term pure vancomycin hydrochloride used herein means vancomycin B hydrochloride identified as the major factor of vancomycin hydrochloride in commercial products.

U.S. Pat. No. 5,854,390 discloses a process for the purification of vancomycin by displacement chromatography on the reverse stationary phase at a pH of the mobile phase between of about 2 to about 10, preferably of about 2 to about 6, while the example presents the mobile phase pH of 3.0, and after applying vancomycin dissolved in the mobile phase onto column, it is displaced by a displacing agent out of the column, the fractions are collected and the combined fractions are lyophilized according to their quality. The particle size of the stationary (reverse) phase is within the range from a few μm to several 100 μm, wherein the example gives the particle size of the stationary phase of 12 μm.

Unexpectedly and surprisingly we have found that the problem known in the prior art is solved by using the new process for the purification of crude vancomycin by displacement chromatography with a stationary phase (reverse phase) of the selected narrow pH of the mobile phase between 3.9 and 4.2 using the particle size of the stationary phase within the range from a few μm to several 100 μm, preferably with the selected particle size of the stationary phase of 5 μm. The stationary phase is octadecyl silica gel.

The process for the purification of crude vancomycin (an aqueous solution with a pH of 3.2) by displacement chromatography on a reverse phase according to the present invention comprises the following steps: a) conditioning the column having a stationary phase with the pH of the mobile phase between 3.9 and 4.2; b) applying crude vancomycin dissolved in the mobile phase onto the column; c) applying a displacing agent to displace vancomycin out of the column and collecting the fractions of the eluate; d) combining and concentrating the fractions of the eluate with purity exceeding 97%; e) adjusting a pH of the vancomycin solution with an aqueous solution of ammonia or alkaline base to a pH between 8.5 to 9.0; f) suspending the separated vancomycin salt from step e) in water and adjusting the medium with hydrochloric acid to a pH of about 3.0 to about 3.5; g) precipitation of vancomycin hydrochloride from the solution with an organic solvent; h) isolation of pure vancomycin hydrochloride.

The column is conditioned with an appropriate mobile phase. The pH of the mobile phase must be moderately acidic because of a lower stability of vancomycin in an alkaline medium and is adjusted with an appropriate acid or an appropriate buffer to the pH of 3.9 to 4.2. The mobile phase can be with water diluted solutions of organic or inorganic acids, such as acetic acid, formic acid, propionic acid, hydrochloric acid, boric acid, phosphoric acid, sulfuric acid, or buffers formed with alkali metal cations, ammonia or amine, such as sodium acetate, ammonium acetate or ammonium phosphate. To achieve a better wetting of the stationary phase, an amount of a few percents (about 3%) of a lower CrC ₄ alcohol, such as methanol, ethanol, acetonitrile or a combination thereof, may be added to the mobile phase. Crude vancomycin dissolved in the mobile phase is loaded onto a column, then a displacing agent to displace vancomycin with the concentration between 5 and 150 mg/ml of the mobile phase, and the fractions of the vancomycin eluate with purity exceeding about 97% are collected. The combined and concentrated fractions of the vancomycin eluate are

concentrated to the concentration of vancomycin between 50 and 150 mg/ml according to their purity quality, an appropriate solvent is then added to the concentrate, such as methanol, the medium is adjusted with an aqueous solution of alkaline base or ammonium hydroxide to a pH between 8.5 to 9.0, and the resulting vancomycin solution is chilled to a temperature between -2O ⁰ C and 5 ⁰ C. The separated vancomycin salt is filtered off, resuspended in water and the medium is adjusted with hydrochloric acid to a pH between 3.0 to 3.5, and vancomycin hydrochloride is precipitated from an aqueous solution in an organic solvent, such as isopropanol. The obtained pure vancomycin hydrochloride is filtered off and dried under vacuum at a temperature to 3O ⁰ C.

We have found that the best results are obtained if a pH of the combined vancomycin eluates with purity exceeding 97% is adjusted to the pH between 8.5 to 9.0 of the vancomycin solution with an aqueous solution of ammonia or alkaline base. As alkali base may be used sodium hydroxide, potassium hydroxide, alkali-earth hydroxides, such as calcium hydroxide and others.

For the preparation of the commercial form of vials the pure vancomycin hydrochloride is lyophilized by the methods known in the prior art.

The purity of vancomycin hydrochloride is determined by High Pressure Liquid Chromatography analytical method as directed in U.S. Pharmacopoeia, United States Pharmacopoeia The National Formulary, 27 ^th revision, 22 (2004), Monograph: "Vancomycin Hydrochloride".

According to the process of the present invention, the pure vancomycin hydrochloride is produced, substantially free of impurities known in commercially available products and in the high total yield. According to the abovementioned HPLC analytical method, the number and the percent portion of impurities were determined in the samples of commercially available vancomycin hydrochloride - "Vancomycin Hydrochloride, USP, manufacturer American Pharmaceutical Partners, Lot: 130373, Exp.: 04/05", vancomycin hydrochloride prepared according to the process disclosed

in US Pat. No. 5,854,390 and in the sample of vancomycin hydrochloride prepared according to the present invention. In the commercially available sample of vancomycin hydrochloride twelve (12) impurities with the assay greater than 0.1% were determined of which five (5) impurities had the assay of each individual impurity above 0.3% as illustrated in the chromatogram in Figure 1. Figure 2 illustrates the chromatogram of the sample of vancomycin hydrochloride prepared as disclosed in US Pat. No. 5,854,390. In contrast to the prior art and the commercially available product, respectively, only two (2) impurities were determined with the assay above 0.1%, of which only one (1) impurity with an assay above 0.3% were determined in the pure vancomycin prepared according to the present invention. This means that the new vancomycin hydrochloride of the present invention contains less than 0.7% of total impurities, that is, only one impurity greater than 0.3%,. Figure 3 illustrates the chromatogram of the sample of vancomycin hydrochloride prepared according to the present invention.

The displacing agent is selected from the group consisting of:

- a higher n-alcohol with C ₄ -Ci ₀ carbon atoms,

- a (di)oxyalcohol (alcohol-ether) - a compound of the type R-O-Y-OH or R-O- Y-O-Y-OH with an ether bond and a hydroxyl end, wherein R is an C _1-I2 alkyl radical and Y is an alkylene group,

- a quaternary ammonium salt with a general formula R ₁ R ₂ RsR ₄ NX, wherein R ₁ , R ₂ , R ₃ and R ₄ being the same or different represent phenyl, benzyl or a Ci _-12 alkyl radical, X is chloride, bromide or iodide e,

- sodium dodecyl sulfate or a hydroxyl derivate thereof.

Preferably, n-pentanol in the form of a solution in the mobile phase is used.

The starting crude and partially purified vancomycin may be prepared by the procedures known in the prior art. Vancomycin is a fermentation product which can be isolated, for example, from a fermentation broth Streptomyces orientalis NRRL 2425 by filtration of the vancomycin fermentative broth and the filtrate is added to a column that contains an adsorption resin that decolorizes and desalts the

δ

vancomycin. The resin is washed, the vancomycin is eluted with an appropriate solvent of low pH of the medium, followed by decolorization of the eluate with active carbon. The vancomycin eluate is then further purified using crystallization at low pH of the medium. The crystallized vancomycin is combined with hydrochloric acid and precipitated in an organic solvent, such as acetone, to form vancomycin hydrochloride.

Preferably, vancomycin from the fermentation broth Streptomyces orientalis is isolated and partially purified as disclosed in U.S. Pat. No. 5,853,720; thereafter, mycelia and other solid substances are separated from the fermentation broth Streptomyces orientalis by microfiltration separation. The resulting permeate is partially purified on an Amberlite XAD 16 acrylate resin column and after eluting with an appropriate solvent, such as acidic methanol (addition of acidic acid), the combined eluates are concentrated by reverse osmosis. The concentrate is then purified with active carbon to produce decolorized concentrate of the (crude) vancomycin which is loaded on a column according to the present invention.

The present invention further relates to the use of the new pure vancomycin hydrochloride for manufacturing the medicinal product for the treatment of bacterial infections and the lyophilized pure vancomycin hydrochloride in sterile form in vials and suitable for injection use.

The present invention is illustrated but in no way limited by the following example:

EXAMPLE

A process for purification of crude vancomycin by displacement chromatography

The stationary phase is an YMC-pack ODS-AQ octadecyl silica gel column (reverse phase) 20 x 250 mm with the particle size of 5 μm (YMC Co. Ltd., Kyoto, Japan). The mobile phase consist of 5 mM aqueous solution of ammonium acetate with the added

3% part of methanol (V/V) and the pH of the medium is adjusted to 4.0 with acetic acid. The displacing agent is 2% solution of n-pentanol in the mobile phase. The sample for loading on the column is 25 ml of an aqueous solution of crude vancomycin (pH adjusted with hydrochloric acid to 3.2, having a concentration of vancomycin 100 mg/ml) to which 9.6 mg of ammonium acetate and 0.75 ml of methanol are added, and the pH is adjusted to 4.0 with an addition of an aqueous solution of ammonia or acetic acid. A flow rate of the mobile phase, the displacing agent and the sample load is 8 ml/min.

The column washed with 250 ml of the mobile phase is loaded the sample, then washed for one minute first with the mobile phase and subsequently the displacing agent is applied. When vancomycin emerge in the eluate, the fractions are collected every 2 minutes. When the concentration of vancomycin in the eluate decrease to the half of the maximum concentration, collecting of fractions are stopped and the column is washed with 250 ml of methanol. The column is then further washed with the mobile phase.

The fractions of the eluate with a chromatographic purity greater than 97.5% (determined by HPLC analytical method according to already mentioned directions from USP 27-NF 22, 2004) are combined, concentrated under vacuum to a concentration of vancomyin of 140 mg/ml. To 12.5 ml of the resulting concentrate first is added 100 ml of methanol, then adjusted a pH of the medium with 1 M aqueous solution of ammonia from pH 8.5 to 9.0, and the resulting solution is chilled to 0 ⁰ C. The separated precipitate is filtered off, washed with 20 ml on 0 ⁰ C chilled methanol and then suspended in 10 ml of water and while stirring a pH of the medium adjusted to 3.2 with 2M hydrochloric acid to effect dissolution of the precipitate. To the resulting solution 50 ml of isopropanol is added, chilled to 0 ⁰ C and filtered off and the resulting vancomycin hydrochloride is dried under vacuum at a temperature of 30 ⁰ C. It is obtained 1.5 g (60% yield) of pure vancomycin B hydrochloride as a white powder and of purity 99.3% (determined by HPLC analytical method according to USP 27-NF 22, 2004) which is stored in polyethylene "AIu-AIu bags" under nitrogen atmosphere.