CH638521A5 - Process for obtaining the antibiotic thienamycin - Google Patents

Description

The invention relates to the isolation of thienamycin from 5 fermentation broths or impure solutions, a substance with antibiotic activity against gram-negative and gram-positive microorganisms; and this using liquid ion exchange systems.

The antibiotic thienamycin is obtained by growing microorganism strains of Streptomyces catt-laya in suitable aqueous nutrient media under controlled conditions. The invention relates to the production of the antibiotic in an essentially pure form.

The method for isolating the antibiotic thiena-15 mycin is described in U.S. Patent 3,950,357. This process works using solid ion exchange resins.

In addition, thienamycin is a hydrophilic, amphoteric compound that cannot be extracted from aqueous solutions by simple organic solvents. Therefore, simple solvent extraction, which is very applicable to the isolation of penicillin and other antibiotics, cannot be used well here.

The method according to the invention works with

25 formation of water-insoluble liquid ion

exchangers dissolved in organic solvents to transfer the thienamycin from the aqueous phase to the organic solvent by the ion exchange mechanism, whereupon the purified thienamycin is transferred from the liquid ion exchange solvent system to aqueous buffers through the mechanism of ion exchange. The use of conventional centrifuge extractors for the ion exchange extraction process leads to extremely rapid mixing and phase separation, 35 which minimizes the time during which the thienamycin is under adverse pH conditions. This leads to higher thiena mycine yields than can be achieved using conventional solid ion exchangers.

40 The invention relates to a method for obtaining and purifying the antibiotic compound thienamycin with the following structural formula:

CH3-CH (OH)

S-CH2-CH2 ~ NH2

COOH

from fermentation broths in which the antibiotic is produced or from solutions that contain partially purified antibiotics. This is achieved by contacting the fermentation broth in which the antibiotic is produced or a solution of partially purified antibiotic with a liquid ion exchanger, dissolved in an organic solvent, for transferring the antibiotic into the liquid ion exchange system (forward extraction) and then contacting the liquid ion exchange system, the contains the antibiotic with an aqueous buffer solution in order to effect the transfer of the antibiotic into the aqueous buffer phase (back-extraction).

With the liquid ion exchange process, the antibiotic is cleaned to a significant extent. The antibiotic of the formula I can also be further purified by desalting and chromatography on polymeric adsorbents.

such as "Amberlite" XAD-1,2 and 4, preferably XAD-2 (manufactured by Rohm and Haas Co., Philadelphia, Penn-55 sylvania), chromatography on strong cation or anion exchange resins such as "Dowex" 50 x 2 ( Well +

Cyclus) or "Dowex" 1x2 (Cl © Cyclus) (manufactured by Dow Chemical Co., Midland, Michigan) and by gel permeation chromatography through polyacrylamide gels. 60 The main advantages of the liquid ion exchange process with regard to the solid ion exchange process are: 1. a higher thienamycin yield and 2. the process can be carried out in a really continuous manner, which results in the economic advantages of continuous operation.

Preferred embodiments are described below:

Thienamycin is used during the fermentation of

suitable aqueous culture media under controlled conditions by a strain of Streptomyces cattleya suitable for producing this compound, such as Streptomyces cattleya NRRL 8057 *. Aqueous media, such as those used to make other antibiotics, are suitable for making thienamycin. Such media contain sources of carbon, nitrogen and inorganic salts which are assimilable by the microorganism.

The preparation and characterization of the antibiotic thienamycin are described in US Pat. No. 3,950,357, the teaching of which is to be encompassed by the present description.

The method according to the invention is based on liquid ion exchangers and an organic solvent. In particular, the ion exchangers are liquid cation exchangers and liquid anion exchangers.

The liquid cation exchangers used in accordance with the invention are preferably of a strongly cationic type, such as dinonylnaphthalene sulfonic acid (DNNS) in the hydrogen *, sodium or other cycles. The term “cycle” used here refers to the special salt form of the liquid ion exchange residue. Other strongly cationic liquid ion exchangers that can be used are didodecylnaphthalene sulfonic acid and its salts. Weaker cationic liquid ion exchangers such as di- (2-ethylhexyl) phosphoric acid can also be used, but the strongly cationic ion exchangers are preferred.

The cation exchanger is used in combination with an organic solvent as the extraction system. The term “organic solvent” is intended to mean an organic solvent or solvent mixture. The organic solvent is said to be one with a moderately high dielectric constant.

The term "moderately high dielectric constant" is intended to mean a dielectric constant from about four to about twenty-four. Examples of such organic solvents with moderately high dielectric constants are straight and branched chain alcohols with four to ten carbon atoms, straight and branched chain ketones with four to eight carbon atoms and straight and branched chain esters with four to ten carbon atoms .

Examples of these alcohols are n-butanol, isobutanol, pentanol, isopentanol, hexanol, heptanol and the like. Examples of these ketones are methyl ethyl ketone, methyl isobutyl ketone and the like. Examples of these esters are ethyl acetate, butyl acetate and the like.

Using a mixed solvent, a solvent with a high dielectric constant can be combined with a solvent with a low dielectric constant to obtain a mixed solvent with the desired dielectric constant.

The term "high dielectric constant" is intended to mean a solvent with a dielectric constant of from about twenty-five to about one hundred. The term "low dielectric constant" is intended to mean under four. A mixture of solvents with moderately high dielectric constants can also be used.

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

• "Filed with Northern Utilization Research and Development Branch of the U.S. Department of Agriculture, Peoria, III.

638521

The liquid anion exchangers are usually salts of strongly anionic materials, such as quaternary ammonium compounds. The liquid anion exchanger can be a salt of tricaprylylmethylammonium, such as the acetate, sulfate, propionate, phosphate, chloride and the like, or it can be in the hydroxyl form. Other types of liquid anion exchangers that can be used are water-insoluble primary, secondary and tertiary amines.

The liquid anion exchanger is also more effective when used with a solvent or solvent mixture that has a moderately high dielectric constant.

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

The anion exchanger is usually used in a solvent solution in which the anion exchanger is about 5 to 30% by volume.

The process for isolating thienamycin is expediently carried out by contacting the acidified (H range from 2.5-4.5) thienamycin-containing broth or solution with the liquid cation exchange system. After the separation of the two liquid phases, the organic phase, which now contains the thienamycin, is back-extracted with an aqueous inorganic buffer such as sodium bicarbonate, ammonium hydroxide, sodium phosphate or potassium phosphate and the like or aqueous pyridine and the thienamycin passes into the aqueous phase. The aqueous phase is then separated from the organic phase.

The last-mentioned aqueous phase containing thienamycin is made alkaline (H range 8.0-11.0) and then brought into intimate contact with the liquid anion exchange system. After separating the two liquid phases, the organic phase, which now contains the thienamycin, is back-extracted with an aqueous buffer such as sodium acetate, potassium acetate, potassium chloride, hydrogen chloride or sodium citrate and the like, and the thienamycin passes into the aqueous phase, which is then separated from the organic phase Phase is separated.

A further purification of the antibiotic can be achieved by desalting and chromatography on polymeric adsorbent resins, such as "Amberlite" XAD-1,2 and 4, preferably XAD-2, chromatography at neutral pH on strong cation exchangers or anion exchangers. exchange resins such as «Dowex» 50 x 2 (Na + Cyclus) or «Dowex» 1 x 2 (Cl © Cyclus) and by gel permeation chromatography using polyacrylamide gels.

The method according to the invention can be applied to fermentation broths or solutions with a wide range of antibiotic concentrations. In general, the higher the antibiotic concentration, the more effective the procedure. For example, the antibiotic concentration can range from 2 mg per liter to about 10,000 mg per liter. However, this is not intended to exclude solutions or broths made with higher concentrations of the antibiotic thienamycin.

Such a procedure includes, for example, the extraction of thienamycin with a strongly acidic liquid cation exchange system at an acidic pH of 2.5-4.5 (forward extraction), the separation of the phases and the subsequent contact of the organic phase with an aqueous back-extracting agent, the separation of the phases and the subsequent contact of the latter aqueous phase with a strongly basic liquid anion

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exchanger system with an alkaline pH of 8.0-11.0 (forward extraction), the phase separation and the subsequent contact of the organic phase with a further back-extraction agent and the subsequent separation of the phases. The latter aqueous solution thus obtained can be further purified by the following procedures: desalting and chromatography on a polymeric absorbent, chromatography on an anion exchange resin of the polystyrene quaternary ammonium type or chromatography on a cation exchange resin, with a buffer or water; gel filtration and chromatography on an adsorbent resin.

The method according to the invention can be carried out in such a way that one of the liquid ion exchangers is used without the other being used. So you can use the liquid anion exchanger with the exclusion of the liquid cation exchanger or you can use the liquid cation exchanger with the exclusion of the liquid anion exchanger.

However, if both ion exchangers are used in succession, the order in which the liquid ion exchangers are used is not critical. So you can use the liquid anion exchanger following the liquid cation exchanger, or the liquid anion exchanger followed by the liquid cation exchanger.

The extractor system used according to the invention can be any system known in the field of the separation of liquids with different densities. It is understood by the person skilled in the art that different centrifuges of different sizes and shapes can be set for optimal results.

example 1

A tube with a lyophilized culture containing a thienamycin-producing Streptomyces cattleya is opened aseptically and the contents in a 250 ml Erlen-Meyer flask with baffles or partitions containing 50 ml of sterile medium B of the following composition , suspended:

Medium B

autolyzed yeast type pH 10 g / 1

Dextrose 10 g / 1

MgS04-7H20 50 mg / 1

KH2PO4 0.182 g / 1

Na2HP04 0.19 g / 1

pH 6.5 before sterilization.

The inoculated flask is shaken at 28 ° C for 24 hours on a rotary shaker at 150 rpm. 3 10 ml portions of the broth treated in medium B for 24 hours are removed aseptically. Each 10 ml portion is immediately mixed with 500 ml Medium B, contained in three 2-1 Erlenmeyer flasks with baffle or partition walls. These three flasks are shaken on a rotary shaker at 150 rpm for 24 hours at 28 ° C.

1500 ml of the 24-hour medium B broths contained in the baffled 2-1 Erlen-Meyer flasks were used directly for inoculating a 756-1 fermentation vessel made of stainless steel, which contained 4671 medium E of the following composition:

Medium E

Glycerin 10 g / 1

Pharmamedia 5 g / 1

CaCL-ÓHiO 0.01 g / 1

"Distillers Solubles"

(soluble stillage) 10 g / 1 CaCC> 3 3 g / 1 polyglycol 2000 2.5 g / 1

pH 7.3 before sterilization.

This container is operated using a stirring speed of 130 rpm and an air flow of 2831 / min for 48 hours at 28 ° C. The fermentation is monitored at 24 hour intervals and listed in the following table:

Old pH

0

6.8

24th

6.8

48

6.5

4541 of the above 48 hour culture broth from the 756-1 stainless steel fermentation vessel are used directly to inoculate a 56701 stainless steel fermentation vessel containing 40821 Medium G of the following composition:

Medium G

Corn steep liquor 15 g / 1

Glycerin 10 g / 1

"Pharmamedia" 5 g / 1

COCI2.6H2O 0.01 g / 1

CaCCb 3 g / 1

Polyglycol 2000 2.5 g / 1

pH 7.3 before sterilization.

This container is operated using a stirring speed of 0.0154 rpm / 1 and an air flow of 0.33981 / 1 for 96 to 100 hours at 25 ° C. The pH of the mixture is adjusted to 6.0 to 7.0.

The 40821 fermentation broth is made using a filter press of 76.2 cm and a filter aid admixture up to an extent of 4% w / v. filtered. An amount of 12 g of the sodium salt of (ethylenedinitrile) tetraacetic acid is then added to the filtrate. The filtrate is cooled to 6 ° C.

The filtered broth is continuously mixed with 2.5N sulfuric acid at about 5 ° C to bring the pH of the broth to 3 using a line mixer. The acidified broth of pH 3 is then fed into a centrifuge extractor at a rate of 2,271 / min and with cold (about 5 ° C) 10% vol / vol dinonylnaphthalene sulfonic acid (DNNS) (initially in the sodium cycle) from pH 2 brought into contact in n-butanol solution, which is fed into the extractor at a rate of 1141 / min. In the extractor, the two solutions are mixed intimately and the cation exchange reaction takes place between Na + -H + ions of the DNNS residue and the ammonium cation form of the thienamycin, which leads to the transfer of the thienaminomycin from the aqueous phase into the solvent phase. The two phases are then carried out by a Podbielniak model D-36 device, which operates at 200 rpm with a centrifugal force up to 2000 g in the extractor,

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effectively separated. The solvent phase containing thienamycin, the enriched DNNS / solvent stream,

is then pumped at a rate of 1141 / min into a second extractor, where it is brought into contact with an aqueous buffer, 6% vol / vol pyridine containing 5 g / 1 dibasic sodium phosphate, for back-extraction. The back extractant is fed into the extractor used for back extraction at a rate of 1141 / min.

About 98% of the thienamycin is extracted from the broth into the DNNS / n-butanol phase in the first extractor and about 95% of the thienamycin is extracted from the DNNS / n-butanol phase into the aqueous buffer using the second extractor. For example, the overall thienamycin yield is approximately 93% from the broth in the aqueous buffer via the liquid cation exchange process.

The aqueous back extractant from the liquid cation exchange process is now mixed with 2.5N sodium hydroxide to bring the pH to 11 using a line mixer. This aqueous stream of about 5 ° C. and pH 11 is fed into a third extractor at a rate of 1141 / min, where it is brought into intimate contact with a liquid anion exchanger, 30% vol / vol "Aliquat 336", Tricaprylyl-methyl-ammonium acetate (acetate cycle) in n-butanol, which is fed in at a rate of 1141 / min. The anion exchange reaction takes place between the negative acetation of the "Aliquat 336" residue and the carboxylate anion form of thienamycin, which leads to a transfer of the thienamycin from the aqueous phase into the solvent phase. The two phases are then effectively separated by the centrifugal forces applied in the extractor. The thienamycin-containing solvent phase, the enriched "ali-quat" / solvent stream, is then pumped into a fourth extractor, where it is brought into contact with an aqueous buffer, 0.40 m potassium acetate (pH 5.0) and again the thienamycin is transferred via anion exchange reactions, this time from the solvent phase to the aqueous buffer phase. The used solvent phase, which contains the «Aliquat 336», is then continuously regenerated to the acetate cycle, using the usual liquid extraction columns.

Through this procedure, about 80 to 85% of the thienamycin from the charged aqueous stream is transferred to the back-extraction buffer via the liquid anion exchange process.

This leads to a substantial purification of the thienamycin. The purity relates to the ratio of the thiena mycin titer to the titer of the total solids dissolved. In the example, the purity was increased thirty times.

If desired, adjust the pH of the extract containing the thienamycin from the liquid anion exchange process to 7-7.2 and concentrate to a 20 volume of 75.71, which is then on a column of 3791 "Amberlite" XAD-2 is applied at a rate of 9.461 / min. The resin is eluted with deionized water at a rate of 18.91 / min and 5681 of an enriched fraction is obtained, it is concentrated to 2.51 for 2 s and carried to 401 one

«Dowex» 50 x 2 (Na + cycle) resin at a rate of 600 ml / min. The resin is then eluted with 600 ml / min of deionized water and 64.41 of an enriched fraction is obtained, which is concentrated to 0.2271 and onto a 30-1 bed of “Bio-Gel” P2 (grain size 0.074 to 0.037 mm), which was previously equilibrated with 0.1 m 2,6-lutidine acetate buffer with pH 7.0. The gel is developed with the same buffer. The enriched fraction is concentrated to 0.1891 and applied to 41 35 "Amberlite" XAD-2 resin. The enriched eluate is concentrated and the concentrate freeze-dried to give about 90% pure thienamycin.

B

Claims (8)

638521 PATENT CLAIMS 1. A method for obtaining the antibiotic thiena-mycin from fermentation broths or solutions containing this antibiotic, characterized in that the broths or solutions with water-insoluble, liquid ion exchanger, dissolved in an organic solvent, for transferring the antibiotic into the brings the liquid ion exchange system into contact by forward extraction and then brings the liquid ion exchange system containing the antibiotic into contact with an aqueous buffer to effect the transfer of the antibiotic into the aqueous buffer phase by back extraction. 2. The method according to claim 1, characterized in that ion exchangers selected from the group of a) liquid cation exchangers, b) liquid anion exchangers, c) liquid cation exchangers, followed by liquid anion exchangers and d) liquid anion exchangers, followed by liquid cation exchangers used. 3. The method according to claim 2, characterized in that dinonyl-naphthalenesulfonic acid or its salts are used as the liquid cation exchange system. 4. The method according to claim 2, characterized in that a salt is used as the liquid anion exchanger, which is selected from the group of tricaprylyl methyl ammonium acetate, tricaprylyl methyl ammonium propionate, tricaprylyl methyl ammonium phosphate and trica-prylyl-methyl-ammonium sulfate. 5. The method according to claim 2, characterized in that a solvent is used with the liquid ion exchangers, which has a moderately high dielectric constant. 6. The method according to claim 5, characterized in that one chooses the solvent from alcohols with four to ten carbon atoms. 7. The method according to claim 5, characterized in that one selects the solvent from ketones with four to eight carbon atoms. 8. The method according to claim 5, characterized in that one selects the solvent from esters with four to ten carbon atoms.