Technical Field

International Patent Classification: C 07 K 9/10; C 07 K 3/20

The present invention relates to a new process for the purification of crude vanco¬ mycin hydrochloride by the use of preparative HPLC (a method of reverse phase dis¬ placement chromatography), whereby the chromatographic purity of the product is improved.

Vancomycin is a tricyclic amphoteric glycopeptide antibiotic in a salt (hydrochloride) form having the empirical formula C ₆₆ H ₇₅ C1 ₂ N ₉ 0 ₂₄ .HC1 and the molecular weight of 1.486 g/mol. The preparation of vancomycin by the fermentation of microorganisms Amycolatopsis orientalis (previously Nocardia orientalis) is described in more detail in US patent 3,067,099. Lyophilized vancomycin hydrochloride has an off-white colour and with water it forms a clear solution having a pH between 2.5 and 4.5.

Vancomycin hydrochloride is particularly used for the initial treatment of serious or severe infections caused by Gram-negative staphylococci resistant against /3-lactam antibiotics as well as in patients who are penicillin-sensitive or do not respond to penicillins and cephalosporines.

Vancomycin hydrochloride is commercially available in oral (solution and capsules/ pulvules) and parenteral (sterile intravenous solution in vials) forms.

Oral use of vancomycin hydrochloride is only allowed in treating staphylococcal en- terocolitis where the parenteral form is not effective. For all other types of indica¬ tions only parenteral use is relevant.

Vancomycin hydrochloride alone or in combination with other aminoglycosides is also useful in treating staphylococcal, streptococcal. enterococcal or diphtherial endocarditis.

To reduce the possibility of side effects a high chromatographic purity of antibiotics is very important in some kinds of their usage, which cannot be achieved by hitherto existing purification processes.

However, it has been achieved by the new process according to the present inven¬ tion, which is more acceptable also from the ecological point ot view due to the use ot non-toxic solvents.

Technical Problem

In the preparation ot vancomycin consisting ot a multistage process involving fer¬ mentation, isolation and various means ot purification, sometimes a product with a more distinctive colour and with an unpleasant odour is obtained and also the yield and purity are reduced.

Since vancomycin represents a very interesting and valuable product, a constant need tor new processes tor effective isolation thereof exists. By the use ot the most hitherto known processes and also by a multiple repetition ot the processes, the poi - tion ot several kinds ot impurities cannot be reduced so as to obtain vancomycin with an improved purity grade.

Therefore the aim ot present invention is to prepare vancomycin hydrochloride ot a new quality class having a considerably greater chromatographic purity than any commercialy available products, which is shown by its whiter colour, and also to ob¬ tain a high yield by using an ecologically irreproachable process.

PnorArt

From patent and other literature a number ot methods tor the preparation ot glycopeptide antibiotics from the fermentation medium as well as tor the purification ot the fermentation products are known, which include precipitation processes using NaOH (EP 323.150, US 5.037,652. JP 5.244,964) or ethanol (US 4,868.285). forma-

tion of phosphates (EP 145,484) or complexes with peptides (US 4.667,024) or imidazole (US 4,868,285), processes for adsorption onto different polvmer resins (US 4,440,753, US 4,845,194, US 4,874.843, US 5,149,784, WO 91/08.300. US 5,258,495 or WO 93/21,207) as well as salting out and precipitation (US 5.235.037).

In these patents chromatography is only used for preparative purposes in US patent 4,667,024 (affinity) and in a part of the process in WO 91/08,300 (ion exchange).

Reverse phase displacement chromatography essentially differs from both types of chromatographies used so far.

The method of displacement chromatography has been known since 1943 and is based on the principle that in a sample the balance between stationary phase (SP) and mobile phase (MP) is shifted the direction of SP. Single components of the sample displace each other like a train and the displacing agent with the greater af¬ finity to SP pushes this train by fractions out of the column. At purification the sub¬ stance is also concentrated. However, due to the lack of effective columns and the complexity of nonlinear chromatography this method was only put into practice in 1981: Cs. Horvath et al., J. Chromatogr., 215 (1981) 295; J. Chromatogr., 330 (1985) 1; J. Chromatogr., 440 (1988) 157. In these articles analytical and preparative separa¬ tion and purification of biologically active peptides and polymyxin antibiotics (polypeptide) by reverse phase chromatography by means of displacement are described. For polymyxins there were used octadecyl silica gel columns (250 x 4.6 mm) with the particle size of 5μ.m, 10% acetonitrile in water as the mobile phase and various tetraalkyl ammonium halides as displacing agents.

Similar columns were used also later in the investigations in the field of displacement chromatography: S.M. Cramer et al., Enzyme Microb. Technol., 11 (1989) 74: Prep. Chromatogr., 1 (1988) 29; J. Chromatogr., 394 (1987) 305; J. Chromatogr.. 439 (1988) 341; J. Chromatogr., 454 (1988) 1 (theoretical optimization); A. Felinger et al, J. Chromatogr., 609 (1992) 35 (theoretical optimization); the mobile phase was methanol in a phosphate buffer and the displacing agent was 2-(2-t-butoxyetoxy)- ethanol (BEE) in acetonitrile and Na-acetate. As samples the different peptides. proteins and antibiotic cephalosporin C were used.

US patent 5,043,423 (27.08.1991) or EP 416,416 disclose a method for purification of some particular low-molecular (below 1000 Daltons) peptides (particularly tuftsin

and its synthetic derivatives) by ion exchange displacement chromatography. wherein the SP is a cation exchange resin, the transport solvent is water or various diluted strong acids and a displacing agent (tπethylene tetraammomum salt) in different con¬ centrations is used.

Commercially available lyophilized vancomycin hydrochloride is in the form ot an off-white powder.

Technical Solution

The manufacturers of the antibiotics are constantly faced with the need to ensure a high yield of medicinal substances with a high chromatographic purity, at as low production costs as possible and, possibly, at a favourable ecological balance This problem is particularly evident at the preparation ot vancomycin due to the con¬ siderable portion of impurities in the crude product, which results in high costs foi its purification. Besides, vancomycin is more polar than peptides due to sugar moieties present in its molecule.

Therefore the present process tor the purification ot vancomycin hydrochloride by displacement chromatography represents an essential improvement over the pπoi art since the portion of impurities m the obtained substance is at least tor one third lower than in the purest hitherto known, commercially available sample, which is evi¬ dent from the very white colour ot the product.

The object ot the present invention is a process tor the purification ot vancomycin hydrochloride by reverse phase displacement chromatography including the follow¬ ing stages:

1. conditioning the column with the selected mobile phase,

2. applying vancomycin hydrochloride dissolved in the mobile phase.

3. applying a displacing agent to displace vancomycin hydrochloride out ot the column and collecting the tractions,

4. analyzing the tractions by analytical HPLC and combining the tractions ac¬ cording to their quality,

5. lyophi zation,

6. regeneration - washing the column with a mixture ot alcohol-water to eluate the displacing agent.

This method is useful for the purification of different peptide substances, particularly more polar glycopeptides having a molecular weight about 5.000 g/mol.

The stationary phase is a reverse phase, which may be natural (silica gel with alkyl chains of different lengths) or a synthetic crosslinked polymer (consisting of styrene and divinylbenzene). The particle size of the stationary phase is within the range from a few μm to several 100 μm.

The pH of the mobile phase must be moderately acidic because of a lower stability of vancomycin hydrochloride in an alkaline medium and is adjusted by the concentra¬ tion of the acid or by the formation of an appropriate buffer. The mobile phase is a diluted organic acid, a halogenated organic or inorganic acid such as formic acid, acetic acid, propionic acid, hydrochloric acid, boric acid, phosphoric acid and sulfuric acid or buffers thereof formed with alkali metal cations, ammonia or amines. To achieve a better wetting of the stationary phase, an amount of a few % of an alcohol, acetonitrile, tetrahydrofuran or of a combination thereof can be added to the mobile phase . After the completed chromatography the stationary phase is regenerated with 20-100% of a lower alcohol in a water solution.

The displacing agent can be: an alcohol with at least a C ₄ -chain (better results are achieved with n-alcohols), 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 alkyl radical and Y is an alkylene group, a quaternary ammonium salt with a general formula R-R ₂ R ₃ R ₄ NX. wherein R-, R ₂ , R-. and R ₄ being the same or different represent phenyl, benzyl or a C,-C _p - alkyl radical, X is chloride, bromide or iodide, sodium dodecyl sulfate or a hydroxy derivative thereof.

Since it is possible to replace the toxic methanol in the mobile phase with the less toxic ethanol without any real difficulties, the present invention represents a con¬ siderable improvement over the prior art also from the ecological point of view due to a simpler removal of the waste solvents.

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

Example

Experimental data tor displacement chromatography are as follows:

The stationary phase was an octadecyl silica gel column 50 x 200 mm (reverse phase ) with a particle size ot 12 μm and the mass ot the stationary phase was 200 g.

The mobile phase (312 ml) with a flow rate ot 45 ml/mm consisted ot 3% methanol or ethanol in acetic acid having a concentration ot 40 mmol/1 so that the pH was 3.0 The mobile phase was displaced by n-pentanol with the concentration ot 200 mmol/1 ot mobile phase. The entering crude vancomycin hydrochloride (25 g) had a con¬ centration ot 80 g/1 ot mobile phase. The deposit was 1 g ot sample per 8 g ot station¬ ary phase or 64 mg ot sample per 1 ml ot stationary phase. The capacity ot the process was 12.5 g ot sample per hour.

The total yield ot the obtained product was 90.6%. The product was separated into 3 tractions with the third traction and, it necessary, the middle traction being repeatedly purified.

Fraction Product Chrom. purity T ₄₀ . Colour Compaπson portion with commercial sample

entering 84.1 % 66 % yellow-brown

95 % 57.8 % 95.5 % 88 % snow white better

92 % 20.8 % 92 % white similar recycled 12.0 % 81 % yellowish

T , is the permeability ot a 5% solution.