The allocation method clavulanic acid

 

(57) Abstract:

The invention relates to biotechnology and relates to a new and improved method of allocation of clavulanic acid from the aqueous culture broth producer clavulanic acid. Mycelium, the major part of proteins and other suspended solids are removed by multistage continuous microfiltration with pH between 5.8 and 6.2 and a temperature of from 20 to 40oC. the Purified broth (aqueous phase) may additionally cleaned by ultrafiltration, and thus purified broth concentrate reverse osmosis. Then it is extracted in countercurrent flow in centrifugal extractors water-immiscible organic solvent at pH 1 and 3. Water-soluble impurities are removed from the organic phase by washing it with water, then dried in a vacuum distillation column to a water content of 0.1%, then concentrated by evaporation, discolor activated carbon, and then clavulanic acid present in the organic phase is isolated and purified by reaction with N,N'-diisopropylethylamine with intermediate diclemente N,N'-diisopropylethylamine that vydelyaemogo product. 12 C.p. f-crystals.

The invention belongs to the field of pharmaceutical industry and relates to a new and improved method of allocation of clavulanic acid and its pharmaceutically acceptable salts from the culture broth of Streptomyces sp. R FERM P 2804.

The need for a new and better way of obtaining pure clavulanic acid and its pharmaceutically acceptable salts, such as potassium clavulanate, by separating them from the culture broth obtained by culturing microorganisms, producing clavulanic acid, and in this way should not be challenging the traditional methods of extraction and chromatographic purification the desired product, there constantly.

Clavulanic acid is a (2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid of the following formula:

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Alkali metal salts and its esters are active inhibitors of beta-lactamases produced by some Gram-positive and Gram-negative microorganisms.

In addition to inhibiting beta-lactamase activity of clavulanic acid and its alkali metal salts give a synergistic effect in combination with beta-Lac the form in galenical preparations to prevent deactivation of beta-lactam antibiotics. Commercial preparations contain more stable potassium salt of clavulanic acid (acid itself is not stable) in combination with amoxicillin trihydrate.

Clavulanic acid is produced by culturing microorganisms, producing clavulanic acid, such as various microorganisms, which represent different strains of the genus Streptomyces, such as S. c1avuligerus NRRL 3585, S. jumoninesis NRRL 5741, S. katsurahamanus IFO 13716 and Streptomyces sp. P6621 FERM P2804.

Water culture broth obtained after cultivation, can be purified and concentrated by conventional methods, including, for example, filtration and chromatographic purification [1], before extraction of the aqueous solution with an organic solvent to obtain a solution of crude clavulanic acid in an organic solvent.

It is known [1] that the salt of clavulanic acid may be obtained by adsorption of the anion application of the filtered broth on the anion-exchange resin and then suirvey electrolyte, the resulting solution bessolevaya and then the solvent is removed. This method can be used to obtain an acceptable yield of the desired substance, although trebuetsya, that restricts the production on a large scale.

Known [2] modified the method of cultivation with the use of strain S. c1avuligerus NRRL 3585, in which the pH of the environment is maintained in the range between 6.3 and 6.7 and, thus, the yield of the desired product increases. Salts of clavulanic acid, such as potassium clavulanate, is obtained by re-scale of application of lithium, resulting in the desired compound is purified.

It is known [3] using tert-butylamine salt of clavulanic acid as a useful intermediate product upon receipt clavulanic acid. This salt is known [4], but only as an ingredient in pharmaceutical compositions.

Known [5] the method of obtaining clavulanic acid by culturing a microorganism S. c1avuligerus. An important improvement of this method was achieved by adding a carbon source such as glycerol in the culture medium during the process, either continuously or intermittently, and it is very important that the concentration of carbon was maintained at sufficiently low, namely below 0.5% (V/o) and in any case not higher than 2%. The examples illustrate that a significant improvement in the sense which iferouane. Found that the concentration of clavulanic acid in the culture broth through 160 h was about 1,400 mg/ml, which is a notable improvement compared to previously known methods.

A further improvement is the new method of purification of clavulanic acid from solution in the form of its lithium salt. However, to achieve higher purity application of lithium add a concentrated solution of other lithium salts such as lithium chloride. Obtained recrystallized clavulanate lithium can be cleaned next, and then possibly converted into other salts, such as potassium clavulanate, known from the literature methods.

Mycelium, proteins and other solids are removed by known methods, such as centrifugation or filtration possible pretreatment of the culture broth selected an aggregating agent to aggregate the mycelium, which makes it possible to facilitate filtering. This separation of the mycelium, proteins and other suspended particles, initially placed in the culture broth in the form of a suspension, is time consuming and requires several working stages.

Require time-consuming ways to remove mycelium, Belkova, as well as obtaining of pure clavulanic acid and its salts can be avoided by using another known method [6, 7].

In General, this method involves three stages, namely the purification of the culture broth from the mycelium, proteins and other solids, purification of clavulanic acid present in the crude form in the broth of the purified filtrate of S. 1avuligerus, using one primary, secondary or tertiary amine, forming a stable intermediate salt of clavulanic acid, after which the main part of related impurities in clavulanic acid is separated, and as the last stage of the transformation of the intermediate amine salts of clavulanic acid (85% purity) in the desired salt of an alkali metal such as potassium clavulanate.

The first stage is described in detail in [6], it is from the aqueous culture broth obtained by culturing a microorganism S. 1avuligerus by physico-chemical methods of coagulation-flocculation to remove the mycelium, proteins and other solids. Obtained in this method, the flakes are large enough and compact, so it is possible to easily sedimentation or separation, it is best DOS is anantaraman by reverse osmosis.

Thereby obtaining the purified culture broth, avoiding traditional cleaning procedures, such as centrifugation, adsorption on activated carbon, filtering with coadjuvante etc.

In all known methods is also necessary (which is different from that described flocculating way) to purified culture broth was purified in a variety of ways of deproteinization and exchange of ions, which leads to significant losses in the final yield of the desired substance. In contrast to well-known methods common in flocculation method is from 85 to 90%.

The described method of coagulation-flocculation from the culture broth of S. c1avuligerus based on the addition of inorganic electrolyte into the culture broth to increase coagulating effect, adding an inorganic coagulant as an initiator of the coagulation process under stirring and the pH of the medium between 6 and 8, adding organic electrolyte, when occulation has already started, and then the separation of the flakes from the culture broth using a rotary sieve or filter, and possibly, if occulation takes place in the presence of water-immiscible Rast is Ariane.

It is known [8] the application of salts of clavulanic acid with organic diamines, such as N,N'-diisopropylethylamine diclofenac, as useful intermediates for the isolation and pure clavulanic acid or its alkali metal salts, such as potassium clavulanate, from an ethyl acetate extract obtained after extraction solvent the resulting aqueous culture broth, which was formed after culturing, which is clavulanic acid.

The present invention is to improve allocation method clavulanic acid from the culture broth obtained by microorganisms, producing clavulanic acid, such as Streptomyces sp. P 6621 FERM P 2804, without requiring time-consuming traditional processes of removal of the mycelium, proteins and other suspended solid particles present in the aqueous culture broth, followed by obtaining salts of high purity, such as clavulanate potassium.

Suitable salts according to the present invention are pharmaceutically acceptable salts of alkali and alkaline earth metals, such as salts of sodium, potassium, calcium is ASEE the invention can be mainly used for cleaning the culture broth, obtained using microorganisms, producing clavulanic acid.

The prior art only methods, which include requiring time-consuming methods of extraction, with the exception of one well-known superior method [6], in which get absolutely clear broth. However, the disadvantage of this method is that to achieve this goal it is necessary to use several reagents, such as inorganic electrolytes, coagulants, organic polyelectrolytes, and occulation, sedimentation or filtration of the culture broth requires a relatively long period of time, which affects the purity of the desired product.

Several possibilities of purifying the culture broth are given on page 2, column 2, lines 22-35 [6], these methods should lead to a significant reduction of the yield clavulanic acid. Next, it is established that the use of several sophisticated techniques in the isolation and purification, such as ultrafiltration and reverse osmosis does not simplify the way, since the use of these processes requires pre-filtering on charcoal or ionoobmennoi resin.

Despite these claims n is known [6] method, and others requiring time-consuming ways to purify water culture broth, known from the literature, which according to the present invention using microfiltration method, through which the mycelium is removed, a large portion of protein (at least 80% present in the broth) and other suspended particles.

For this purpose use a multistage device for continuous microfiltration, which makes it possible to implement a way to separate the mycelium and the aqueous filtrate for time delays less than half an hour, and the device consists of several (five) series-connected independent segments (filtration loops). Each segment has its own circulation pump, which allows the culture broth with the desired speed (5-8 m/s) through the channels of the ceramic filter element having a pore size of 0.05 μm. Microfiltration process takes place at a temperature between about 20 and 40o(The temperature should not exceed 40oC), the tangential speed is regulated so that the solid fraction remained molecules with a molecular weight of more than 30,000. This is achieved by removing approximately 80-90% of bristlebane clavulanic acid in the combined filtrate. By this method the aqueous culture broth by microfiltration more than 95% of the clavulanic acid remains in the aqueous phase, which exceeds the results flocculation method [6] and represents another improvement provided by the present invention.

After filtration, the filtrate can be purified by ultrafiltration. The purpose of this purification is the separation of the greater part of the remaining protein impurities and other related impurities having a molecular weight greater than that of clavulanic acid. Thus impurities that could precipitate followed by extraction with water-immiscible organic solvent, successfully removed, after which the color of the water filtrate obtained after purification by microfiltration, will be substantially reduced and the purity of the desired product even better.

In ultrafiltration device using a polymer membrane having a high degree of resolution, which is about 20,000 daltons (between 10000 and 30000 daltons). The method is carried out in continuous mode, so that the delay time was how short as possible, and two ultrafiltration device connected posledovatelnoy phase is reduced to less than 5% by the addition of pure water for washing, and by removing the retaining medium (aqueous phase) obtained in the process of ultrafiltration, reverse current.

The combined aqueous filtrates are then concentrated in the device for reverse osmosis at a temperature of approximately room temperature to about 1/5 of the initial volume with obtaining a concentrated aqueous phase crude clavulanic acid and then the resulting concentrate (holding environment) directly extracted at a temperature of between 15 and 25o(Extraction can also be carried out at temperatures below 15oC) a water-immiscible organic solvent, such as ethyl acetate, in an acid medium at a pH in the range from 1 to 3, which fail by adding an inorganic acid such as sulfuric acid. In addition to ethyl acetate is also possible to use other water-immiscible organic solvents such as methyl acetate, methyl isobutyl ketone or n-butyl alcohol.

As microfiltration process is allowed to remove the mycelium and the main part of the present proteins, direct extraction of purified and concentrated aqueous culture broth with a suitable water-immiscible organic solvent, such as EMERAUDE in the known methods, and it is possible to avoid the use of additional reagents, which are used in the method of coagulation-flocculation for cleaning the culture broth. Thus, in addition to the above improvement of the method according to the invention also reduces the cost of cleaning broth. To avoid denaturation of the remaining proteins in the concentrate of the aqueous phase due to the interaction with an organic solvent or hydrochloric acid in the extraction process of crude clavulanic acid from the aqueous phase to the organic phase, it is better to perform the extraction in series centrifugal extraction apparatus, when one of them, namely Samooborona centrifugal separator, simultaneously and in a continuous process removes the separated proteins.

The resulting crude extract of hydrochloric acid in a water-immiscible organic solvent can be water-soluble impurities, such as different products of the decomposition of clavulanic acid, which are more polar than the clavulanic acid, therefore, water-soluble impurities are removed by washing the combined organic phases with water. Thereby obtaining a purified extract of clavulanate from the organic phase and cleaned in a known manner [8]. The best way to highlight clavulanic acid according to [8] is the interaction of an ethyl acetate extract of clavulanic acid with N,N'-diisopropylethylamine at a temperature approximately equal to room temperature and subsequent conversion of the obtained intermediate diclemente N,N'-diisopropylethylamine 2-ethylhexanoate potassium in an aqueous solution of isopropanol at room temperature with the receipt of application of potassium, which emit in a very pure form.

The authors of the present invention, it was found that the intermediate diclemente N,N'-diisopropylethylamine is best done so that in the reaction between the water-immiscible organic phase, such as an ethyl acetate extract clavulanic acid, and N,N'-diisopropylethylamine to use the organic phase, which completely removed the water, as even a small amount of water may prevent intermediate salt, due to the fact that the selected salt will dissolve into the present in the organic phase and water can be obtained by-products, which will impede drying.

If the water is completely removed from the organic phase, the stability ardneh solutions in an environment with an acidic pH in the extraction process is very small. Therefore, drying of the organic phase, such as an ethyl acetate extract clavulanic acid, used drying in a distillation column (on the principle of fractional distillation in a vacuum because of the low stability of the intermediate salt at high temperatures. The essential feature of this method is that the organic phase, such as ethyl acetate, and water form an azeotrope having a lower boiling point, and thus the organic phase, such as an ethyl acetate extract is thus fully dried. Thus organic phase, such as an ethyl acetate extracts, always contains water in an amount less than 0.1 vol %, on average from 0.03 to 0.05% by volume. Then completely dehydrated organic phase, such as an ethyl acetate extract of clavulanic acid in a very short time delay is concentrated by evaporation to 1/20 of the original volume for subsequent interaction with N,N'-diisopropylethylamine.

Subsequent reaction of diclofenaco N,N'-diisopropylethylamine and 2-ethylhexanoates potassium to receive application of potassium high purity can be carried out in a known manner [8] and is best carried out further Examples, not having a restrictive nature.

Example 1

The continuous process of obtaining an ethyl acetate concentrated extract containing crude clavulanic acid.

Water culture broth (10000 l), obtained by culturing the microorganism Streptomyces sp. P6621 FERM P 2804 (concentration of clavulanic acid to 3580 mg/l) are added to a 33% increase aqueous solution (5 l) sulfuric acid in a vessel with a capacity of 50 m3under stirring and cooling so that the value of pH of the medium is maintained between about 5.8 and 6.2. Then the broth continuously at a flow rate of 1200 l/h serves on microfiltration device, consisting of 5 series-connected segments. Each segment has its own circulation pump to ensure the speed of passage of the culture broth through the channels of the ceramic filter element with a pore size of 0.05 μm at the level of 8 m/sec. By microfiltration process, provided that the temperature does not exceed 40oWith, the mycelium is removed and the main part of proteins and other suspended solids.

The separated solid is washed with water at a flow rate of 300 l/h and then the combined filtrate (penetrated Wednesday) after ICRI the medium is concentrated to 1/5 of the initial volume.

To the concentrate (holding environment), obtained after reverse osmosis with a flow rate of 300 l/h, add 33% aqueous solution (4 l/h) sulfuric acid so that the pH of the medium is maintained between 1.5 and 2.0, then a flow rate of 900 l/h add ethyl acetate for extraction of acidic holding environment at room temperature in a counter in a series of five centrifugal extraction apparatus, and the second Samooborona centrifugal separator are removed simultaneously remaining still proteins.

United an ethyl acetate extract of the series centrifugal extraction apparatus is washed in the first centrifugal extraction apparatus demineralized water with a flow rate of 30 l/h and thus remove the remaining are still water-soluble impurities.

An ethyl acetate extract at a flow rate of 900 l/h dried in a vacuum at a temperature of 30oWith in a distillation column, that is, the water content of 0.03% by volume, then the extract is evaporated in a thin layer evaporator under vacuum at a temperature of 30oWith up to 1/20 of the original volume. The concentrated an ethyl acetate extract (concentration of crude clavulanic Kies stirred for 30 min and then filtered coal from the concentrated suspension of an ethyl acetate extract at a pressure filter under nitrogen pressure of 1 bar with getting dry concentrate (45 l) of an ethyl acetate extract, containing crude clavulanic acid.

Example 2

Getting diclemente N,N'-diisopropylethylamine

To the dry concentrate (45 l) of an ethyl acetate extract obtained in a continuous process in Example 1 (content of clavulanic acid to 50 g/l) for 5 minutes with thorough stirring at a temperature of 25oWith added N, N'-diisopropylethylamine (1.4 l). The resulting suspension is filtered, the obtained crystals resuspended in acetone (45 l) and under stirring and cooling the slurry to a temperature below 10oWith separate crystals of the desired substance, filtered off, washed with acetone and dried in a vacuum at a temperature of 30oC. Get the crystals diclemente N,N'-diisopropylethylamine (3.3 kg; the content of clavulanic acid to 60%).

Example 3

Obtaining application of potassium

Diclofenac N,N'-diisopropylethylamine (3.3 kg) from Example 2 is dissolved in a mixture of isopropanol/water (82,5 l; the proportion of water to 1.5%) and to the resulting solution for 30 min under stirring at room temperature, add activated charcoal (1.5 kg) and 2-ethylhexanoate, potassium (0,5 l; 2 M). Then the coal and the precipitate is filtered off. To polycentrality potassium (2 M) in isopropanol. The resulting slurry is then stirred while cooling to a temperature of between 0 and 5oWith another 2 h, and then the separated crystals are filtered off, washed with isopropanol and acetone and dried in a vacuum at a temperature of 30oC. Receive clavulanate potassium (2 kg; USP purity, content of clavulanic acid 80.6% of defined ghvd).

Sources of information

1. GB 1508977.

2. GB 1543563.

3. EP-A-0026044.

4. BE 862211.

5. EP-IN-0182522.

6. EP-A-0385552.

7. EP-A-0387178.

8. EP-A-0562583.

1. The allocation method clavulanic acid, including the removal from the aqueous culture broth and mycelium, the main part of proteins and other suspended solid particles, followed by extraction clavulanic acid water-immiscible organic solvent and isolation of the target product from the organic phase, characterized in that the removal is carried out by a multi-stage continuous microfiltration at a temperature of 20-40oC.

2. The method according to p. 1, wherein the microfiltration lead through filtration element having a pore size of 0.05 μm.

3. The method according to any of paragraphs.1 and 2, characterized in that m is I, the extraction is carried out at pH 1-3 using centrifugal extraction device with a counter.

5. The method according to p. 4, characterized in that the extraction was carried out sequentially in series centrifugal extraction machines.

6. The method according to any of paragraphs.1, 4 and 5, characterized in that the organic solvent is chosen from the group comprising ethyl acetate, methyl acetate, methyl isobutyl ketone and n-butanol.

7. The method according to any of paragraphs.1 and 4-6, characterized in that the organic solvent is an acetate.

8. The method according to any of paragraphs.1-3, characterized in that after filtration, the obtained filtrate is additionally cleaned by ultrafiltration.

9. The method according to p. 8, characterized in that the ultrafiltration carried out continuously using a semi-permeable membrane having a degree of resolution between 10 and 30 kDa.

10. The method according to any of paragraphs.1-9, characterized in that before carry out extraction stage removal of water by reverse osmosis.

11. The method according to any of paragraphs.1-10, characterized in that the selection of the target product from the organic phase comprises drying in a vacuum using a distillation column.13. The method according to any of paragraphs.1-12, characterized in that the selection of the target product from the organic phase includes the implementation of the interaction of diclofenaco N,N'-diisopropylethylamine 2-ethylhexanoate potassium obtaining pure application of potassium.

 

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< / BR>
where R1, R2, R3and R4independently represent hydrogen, a straight or branched alkyl WITH1-C8hydroxyalkyl2-C4or the group NR1R2and NR3R4together denote a heterocyclic group containing 3 to 6 methylene groups, possibly substituted by oxygen, sulfur or aminogroups, R5denotes hydrogen or methyl, n is an integer 1 to 3; the allocation of the resulting amine salt of clavulanic acid; and converting the amine salt in the clavulanic acid or its pharmaceutically acceptable salt or ester

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