DK144097B - PROCEDURE FOR THE RECOVERY OF ANTIBIOTIC SUBSTANCE THIENAMYCIN - Google Patents

Description

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(19) DENMARK

H (12) PUBLICATION <N> 144Q97B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 67 ^ / 78 (51) jnt.CI.3 C 12 P 17/18 (22) Filing day 15 · bold> · 1978 C 07 D 487/04 (24) Running day 15 Feb. 1978 (41) Aim. available Sep 2 1978 (44) Posted 7 Dec. 1981 (86) International application no.

(86) International Filing Day - (85) Continuation Day “(62) Application for Stock”

(30) Priority 1 Mar 1977, 773551 *, US

(71) Applicant MERCK & CO. INC., Rahway, US.

(72) Inventor Rathin Datta, US: George Thomas Wildmsn, US, (74) Hofman-Bang See Engineering Company Boutard.

———— ^^^^ - ^ - 111 .............. —...................

(54) Method for extracting the antibiotic thienaraycin.

The present invention relates to a method for recovering the antibiotic thienamycin.

Thienamycin is obtained by growing a microorganism of the species Strep-tomyces cattleya in a suitable nutrient medium under controlled? BE-conditions.

It is known from US Patent No. 3,950,357 to isolate thiena- mycine using solid ion exchange resins.

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Thienamycin having the formula 3 CH3-CH (OH) -> -S-CH? -CHp-NH

J

C00H

is a hydrophilic amphoteric compound which cannot be extracted from aqueous solutions by simple organic solvents.

Therefore, a simple solvent extraction known from the isolation of penicillin and other antibiotics cannot be used in the present case.

The present invention is based on the recognition that water-insoluble liquid ion exchangers dissolved in suitable organic solvents can be used to transfer the thienamycin by an ion exchange mechanism from the aqueous phase to the organic solvent followed by transfer of the purified thienamycin liquid ion exchange / solvent mixture to a suitable aqueous buffer, again by ion exchange. The use of conventional centrifugal extractors for the ion exchange extraction process has led to an extremely rapid mixing and phase separation, thereby reducing the time at which the thienamycin is subjected to harmful pH values. This procedure provides improved yields of thienamycin over the use of conventional solid ion exchangers.

Accordingly, the method of the invention is peculiar to the characterizing part of claim 1.

Substantial purification of the antibiotic occurs by the liquid ion exchange process. The antibiotic of the above formula I may also be further purified by desalination and chromatography on polymeric adsorbents such as Amberlite XAD-1, 2 and 4, preferably XAD-2 (manufactured by Rohm and Haas Co., Philadelphia, Pennsylvania), chromatography on highly cationic or anionic ion exchange resins, such as Dowex 50x2 (Na + form) or Dowex 1x2 (Cl "form) (manufactured by Dow Chemical Co., Midland, Michigan) and by gel filtration chromatography through polyacrylamide gels .

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The main advantage of the liquid ion exchange process over the conventional solid ion exchange process is the following: 1) greater yield of thienamycin and 2) the process can be carried out completely continuously, giving the advantages characteristic of continuous operation.

Thienamycin is prepared, as mentioned, by aerobic fermentation of suitable aqueous foods under controlled conditions, using as a microorganism a strain of Streptomyces cattleya, such as the microorganism registered under the designation NRRL 8057. Fermentation is carried out in the usual aqueous media commonly used for manufacture of antibiotics. Such media contain sources of carbon, nitrogen and inorganic salts that can be assimilated by the microorganism.

The preparation and characterization of thienamycin is described in U.S. Patent No. 5,950,357.

Liquid cation exchange and liquid anion exchangers can be used in the process according to the invention.

Examples of liquid cation exchangers are the highly cationic types, such as dinonylnaphthalenesulfonic acid (DNNS) in hydrogen, sodium or other form. The term "form" refers to the particular salt form of the liquid ion exchanger. An example of another strong ionic liquid ion exchanger is dodecyl naphthalene sulfonic acid and its salts. Weaker cationic liquid ion exchangers such as di- (2-ethylhexyl) phosphoric acid may also be used, but the highly cationic ion exchangers are preferred.

The cation exchangers are used in combination with an organic solvent or a mixture thereof as an extractant. The organic solvent should have a moderately high dielectric constant, such as between 4 and 24. Typical examples of such organic solvents having a moderately high dielectric constant are straight or branched alcohols of 4-10 carbon atoms, straight or branched ketones of 4-8 carbon atoms, and straight or branched esters of 4-10 carbon atoms.

Typical examples of said alcohols are n-butanol, iso-butanol, pentanol, isopentanol, hexanol and heptanol. Typical examples of the ketones are methyl ethyl ketone and methyl isobutyl ketone. Examples of suitable esters are ethyl acetate and butyl acetate.

When using a mixture of solvents, a solvent with a high dielectric constant can be combined with a solvent with a low dielectric constant to obtain a mixture with the desired dielectric constant. By the term "high dielectric constant" is meant a solvent having a dielectric constant between 24 and 100. By the term "low dielectric constant" is meant less than 4.

However, a mixture of solvents with moderately high dielectric constants can also be used.

The cation exchanger is usually used at a concentration of 2 to 15% by volume. in the solvent. The liquid cation exchange solution is then adjusted to a pH between 1 and 4 with a suitable aqueous buffer.

The liquid anion exchangers are usually salts of highly anionic materials such as quaternary ammonium compounds. The liquid anion exchanger may be a tricaprylyl methyl ammonium salt such as acetate, sulfate, propionate, phosphate or chloride, or may be hydroxyl form. Other types of liquid anion exchangers which may be used are water-insoluble primary, secondary and tertiary amines.

The liquid anion exchanger is also used with a solvent or mixture thereof with a moderately high dielectric constant.

The above-described solvents and mixtures thereof which can be used with the cation exchanger can also be used for the anion exchanger.

The anion exchanger is usually used at a concentration of 5 to 50% by volume. in the solution.

The present process can be used in connection with fermentation liquids or solutions with widely varying concentrations of the antibiotic. In general, the process will be most effective at the high concentrations. Eg. the concentration of the antibiotic can be between 2 mg per liter and 10,000 mg per liter. liter. However, it is not intended to exclude solutions or fermentation liquids prepared with a higher concentration of the thienamycin antibiotic.

According to one embodiment, the thienamycin can be extracted with a highly acidic liquid cation exchanger at acidic pH, namely from 2.5 to 4.5 (forward extraction), separation of the phases and then treatment of the organic phase with an aqueous back extractant, separation of the phases followed by treatment of the latter aqueous phase with a highly basic liquid anion exchange mixture at alkaline pH, namely from 8.0 to 11.0 (forward extraction), separation of the phases and subsequent treatment of the organic phase with another aqueous back-extractant and then phase separation. The latter solution thus obtained can be further purified by the following process: desalination and chromatography on a polymeric adsorbent, chromatography on an anion exchange resin of the polystyrene quaternary ammonium type or chromatography on an anion exchange resin with a buffer or water, gel filtration and chromatography on an adsorbent resin .

The process of the invention can be carried out using any of the liquid ion exchangers without the use of the others. Thus, the liquid anion exchanger can be used to close the liquid cation exchanger, or the liquid cation exchanger can be used to exclude the liquid anion exchanger.

If both liquid ion exchangers are used sequentially, it is not critical which one of the ion exchangers is used. Thus, the liquid cation exchanger followed by the liquid anion exchanger can be used, or the liquid anion exchanger can be used followed by the liquid cation exchanger.

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Any extraction device known per se can be used for extraction. Thus, centrifuges of different types and sizes can be used.

The method according to the invention will now be further illustrated by way of example.

EXAMPLE

A tube of lyophilized culture containing a thienamycin-producing strain of Streptomyces cattleya is opened aseptically and the contents are suspended in a 250 ml aerated conical flask containing 50 ml of sterile medium B having the following composition:

Medium B

Autolysed yeast of type pH 10 g / l

Glucose 10 g / l

Mg.SO47H20 50 mg / l KH2 PO4 0.182 g / l

Na2HPO4 0.19 g / l pH 6.5 before sterilization

The inoculated flask is shaken at 28 ° C and 150 rpm in a rotary shaker for 24 hours. Three 10 ml aliquots of Medium B are removed aseptically after 24 hours of culture. Each 10 ml aliquots are immediately mixed with 500 ml of medium B in three 2 liter aerated conical flasks. These seed flasks are shaken at 28 ° C in a rotary shaker at 150 rpm. for 24 hours.

I500 ml of Medium B is fermented for 24 hours and the contents of the tapered 2 liter flasks are used immediately for inoculation of a 756 liter stainless steel fermenter containing 467 liters of Medium E of the following composition: 7 144097

Medium E

Glycerol 10 g / L

Pharmamedia 5 g / l

CaCl2-6H20 0.01 g / l

Distilled solution 10 g / l

CaCO 3 3 g / L

Polyglycol 2000 2.5 g / l pH 7.3 before sterilization

This tank is operated at 28 ° C with a shaking speed of 130 rpm. and a flow of air of 283 liters per minute for 48 hours. The pH of the fermentation liquid is controlled at a 24 hour interval according to the following table:

Age pH

0 6.8 24 6.8 48 6.5 454 liters of the above 48 hour fermentation liquid in the 756 liter stainless steel container are immediately used for grafting a 5670 liter stainless steel fermentation container containing 4082 liters of Medium G with the following composition:

Medium G

Maize casting liquid 15 g / l

Glycerol 10 g / L

Pharmamedia 5 g / l

CaCl 2 · 6H 2 O 0.01 g / l

CaCO 3 3 g / L

Polyglycol 2000 2.5 g / l pH 7.3 before sterilization

This tank is operated at 25 ° C and a speed of 0.0154 rpm and an air flow of 0.34 l / liter for 96 to 100 hours. The pH of the fermentation liquid is adjusted to 6.0 to 7.0.

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The 4082 liters of fermentation liquid is filtered through a 75 cm filter press, adding a filtration aid in the amount of 4% w / v. To the filtrate is added 12 g (ethylenedinitrile) -tetraacetic acid, sodium salt. The filtrate is cooled to 6 ° C.

The filtered liquid at 5 ° C is continuously mixed with 2.5 normal sulfuric acid to obtain a pH of 3 »using a built-in mixer. The liquid, adjusted to a pH of 3, is then fed at a feed rate of 228 liters per liter. per minute to a centrifugal extractor, where it is treated with cold (about 5 ° C) 10% v / v dinonylnaphthalenesulfonic acid (DrØS) (primarily in sodium form) at a pH of 2 in n-butanol solution fed extractor at a rate of 114 liters per liter. minute. In the extractor, the two solutions are thoroughly mixed and the cation exchanger reacts between the Na + and H + ions of the DMS moiety and the ammonium cation form of thienamycin so that the thienamycin is transferred from the aqueous phase to the solvent phase. The two phases are then effectively separated with a Podbielniak model D-36, which is operated at 200 rpm. for developing a centrifugal force up to 2000 G in the extractor. The thienamycin-containing solvent phase, the enriched DIMS / solvent stream, is then pumped at a rate of 114 liters per liter. to another extractor, where it is treated with an aqueous buffer, 6% v / v pyridine containing 5 g / l dibasic sodium phosphate, for back extraction. The back extraction operates at a feed rate of 114 liters per minute. minute to the extractor.

Ca. 98% of the thienamycin is extracted from the liquid in the DNNS / n-butanol phase of the first extractor, and ca. 95% of the thienamycin is extracted from the DMS / n-butanol phase to the aqueous buffer using the second extractor. Thus, the total thiena-mycine yield is approx. 93% from the liquid to the aqueous buffer via the liquid cation exchange process.

The aqueous back extract from the liquid cation exchange process is then mixed with 2.5 normal sodium hydroxide to adjust a pH of 11 by means of a built-in mixer. This aqueous stream, ca. 5 ° C and pH = 11, are fed at a rate of 30 liters per day. to a third extractor, where it is treated intimately with a liquid ion exchanger 30% v / v "Aliquat 336", tri-caprylyl methylammonium acetate (acetate form), in n-butanol, which is fed at 114 liters per minute. minute. The anion exchange reaction occurs between the negative acetation in the "Aliquat 336" part and the carboxylate anion form of thienamycin to obtain a transfer of thienamycin from the aqueous phase to the solvent phase. The two phases are then effectively separated by the centrifugal forces acting in the extractor. The thienamycin containing the solvent phase, the rich aliquat / solvent stream, is then pumped to a fourth extractor where it is treated with an aqueous buffer, 0.40 molar potassium acetate (pH = 5.0) and again, via the anion exchange reactions, the thienamycin is transferred, this time from the solvent phase. to the aqueous buffer phase. The spent solvent phase containing "Aliquat 336" is then continuously regenerated to acetate cycle using conventional liquid extraction columns.

In the described procedure, approx. 80 to 85% of the thiena-mycine from the aqueous feed stream to the back extraction buffer via the liquid anion exchange process.

Substantial purification of the thienamycin occurs. The purity refers to the ratio of thienamycin titre to titre for total dissolved solids. In the example, purity was increased by a factor of 30.

If desired, the thienamycin-containing extract from the liquid anion exchange process is adjusted to a pH of 7-7.2 and concentrated to a volume of 76 liters, which is then applied to a 378 liter column of Amberlite XAD-2 at a rate of 9 , 5 l / min. The resin is eluted with deionized water at 19 l / min, and a fraction of 570 liters is collected and evaporated to 2.5 liters, which is then applied to 40 liters of Dowex 50 x 2 (Na + form) resin at a rate of 600 ml / min. .

The resin is then eluted at 600 ml / min. with deionized water and a fraction of 64 liters is collected and evaporated to 0.23 liters. This concentrate is applied to a 30-liter layer of Bio-Gel P-2 (200-400 mesh) which is preset to equilibrium with 0.1M 2,6-lutidine acetate buffer at pH = 7.0. The gel is then developed with the same buffer. The enriched fraction included