GB1568924A - Thienamycin recovery - Google Patents

Abstract

A thienamycin-containing fermentation broth or solution is brought into contact with a liquid ion exchanger which is dissolved in an organic solvent and is insoluble in water. In this process, the antibiotic is transferred, by forward extraction, into the liquid ion exchanger system. The liquid ion exchanger system, containing the antibiotic, is then brought into contact with an aqueous buffer. During this process, the antibiotic is transferred, by back extraction, into the aqueous buffer phase. The process is simple to carry out and the antibiotic thienamycin accrues, as the end product, in high yield and in pure form. Thienamycin exhibits antibiotic activity against Gram-negative and Gram-positive microorganisms.

Description

(54) THIENAMYCIN RECOVERY (71) We, MERCK & CO. INC., a corporation duly organized and existing under the laws of the State of New Jersey, United States of America, of Rahway, New Jersey, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed to be particularly described in and by the following statement: The antibiotic thienamycin is obtained during the aerobic fermentation of suitable aqueous nutrient media, under controlled conditions, by a strain of Streptomyces cattleya capable of producing said compound such as Streptomyces cattleya NRRL 8057. Aqueous media, such as those used for the production of other antibiotics, are suitable for producing thienamycin.

Such media contain sources of carbon, nitrogen and inorganic salts assimilable by the microorganism.

The present invention is directed to the methods for recovering the antibiotic in substantially pure form. In view of the provisions of Section 9 of the Patents Act 1949, attention is directed to our prior patent No. 1 498 087. A process for the isolation of the antibiotic thienamycin is reported, and the antibiotic is characterized, in U.S. Patent No. 3 950 357.

This process utilizes solid resin ion exchangers. However, thienamycin is a hydrophilic, amphoteric compound and it cannot be extracted from aqueous solutions by simple organic solvents. Hence, simple solvent extraction, which is very suitable for the isolation of penicillin and other antibiotics, cannot be readily applied here.

The present invention provides a method of recovering thienamycin from fermentation broths or solutions containing it that comprises transferring the antibiotic into a solution of a water-insoluble liquid ion exchanger in an organic solvent (forward extraction) and then bringing the liquid ion-exchange system into contact with an aqueous buffer solution to transfer the antibiotic into the aqueous buffer phase (back extraction), in which the ion exchanger is (a) a liquid cation exchanger; (b) a liquid anion exchanger; (c) a liquid cation exchanger followed by a liquid anion exchanger; or (d) a liquid anion exchanger followed by a liquid cation exchanger. The use of conventional centrifugal extractors for the ion-exchange extraction process leads to extremely fast mixing and phase separations thereby minimizing the time that thienamycin is under adverse pH conditions. This results in higher thienamycin recoveries than obtained by the use of conventional solid ion exchangers. The starting material for the process of the present invention will usually be a fermentation broth in which the antibiotic has been produced or a solution containing partially purified antibiotic: the liquid ion exchangers used can be liquid cation exchangers or liquid anion exchangers.

Substantial purification of the antibiotic occurs in the liquid ion-exchange processes. It may be further purified by desalting and chromatography on polymeric adsorbants such as that sold under the trade mark Amberlite XAD-1, 2 and 4, preferably XAD-2 (manufactured by Rohm and Haas Co., Philadelphia, Pennsylvania), chromatography on strong (cation or anion)-exchange resins such as that sold under the trade mark Dowex 50 x 2 (Na+ cycle) or Dowex 1 x 2 (Cl cycle) (manufactured by Dow Chemical Co., Midland, Michigan) and by gelpermeation chromatography through polyacrylamide gels.

The principal advantages of the liquid ion-exchange process over the conventional solid ion-exchange process are (1) the recovery of thienamycin can be improved; and (2) the process can be operated in a truly continuous mode, thus giving the economic advantages of a continuous operation.

The liquid cation exchangers utilized in this invention are preferably those of the strong cationic variety such as dinonylnaphthalene -sulfonic acid (DNNS) in the hydrogen, sodium or other cycles. The term "cycle" refers to the particular salt form of the liquid ion exchanger residue. Other strong cationic liquid ion-exchangers that can be used are didodecylnaphthalenesulfonic acid and its salts. Weaker cationic liquid ion exchangers, such as di-(2ethylhexyl) phosphoric acid, may also be used; however, the strong cationic ion exchangers are preferred.

The cation exchanger is usually utilized in combination with an organic solvent as the extraction system. By the term "organic solvent" is means an organic solvent or solvent mixture. The organic solvent preferably has a moderately high dielectric constant, by which term is meant a dielectric constant from four to twenty-four. Representative of such organic solvents having moderately high dielectric constants are straight and branched chain alcohols having from four to ten carbon atoms, straight and branched chain ketones having from four to eight carbon atoms and straight and branched chain esters having from four to ten carbon atoms.

Representative of the said alcohols are n-butanol, isobutanol, pentanol, isopentanol, hexanol and heptanol; representative of the said ketones are methyl ethyl ketone and methyl isobutyl ketone, and representative of said esters are ethyl acetate and butyl acetate.

When a solvent mixture is used, one solvent with a high dielectric constant may be combined with a solvent having a low dielectric constant in order to obtain a solvent mixture having the desired dielectric constant.

By the term "high dielectric constant" is meant a dielectric constant from twenty-five to one hundred. By the term "low dielectric constant" is means a dielectric constant less than four. Also, a mixture of solvents having moderately high dielectric constants may be used.

The cation exchanger usually makes up 2 to 15% by volume of the solution. The liquid cation exchange solution is then adjusted to a pH between 1 to 4 with a suitable aqueous buffer.

The liquid anion exchangers are usually salts of strong anionic materials such as quaternary ammonium compounds. The liquid anion exchanger can be a tricaprylyl methyl ammonium salt such as the acetate, sulfate, propionate, phosphate or chloride, or the hydroxyl form.

Other types of liquid anion exchangers that may 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 having a moderately high dielectric constant.

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

The anion exchanger usually makes up from 5 to 30 % by volume of the solvent solution.

The process for thienamycin isolation is carried out by contacting the acidified (pH range from 2.5-4.5) thienamycin-containing broth or solution with the liquid cation-exchange system. After 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, or with aqueous pyridine, and the thienamycin transfers to the aqueous phase, which is then separated from the organic phase.

The latter thienamycin-containing aqueous phase is made alkaline (pH range from 8.011.0) and then intimately contacted with the liquid anion-exchange system. After separation of the two liquid phases, the organic phase, which now contains the thienamycin, is backextracted with an aqueous buffer such as sodium acetate, potassium acetate, potassium chloride, hydrogen chloride or sodium citrate and the thienamycin transfers to the aqueous phase which is then separated from the organic phase.

Further purification of the antibiotic may be obtained by desalting and chromatography on polymeric adsorbent resins like Amberlite XAD- 1, 2 and 4, preferably XAD-2, chromatography at neutral pH on strong cation exchange or anion exchange resins such as Dowex 50 x 2 (Na+ cycle) or Dowex 1 x 2 (Cle cycle) and by gel permeation chromatography using polyacrylamide gels.

The process described herein can be utilized with fermentation broths or solutions over a wide range of antibiotic concentrations. In general, the higher the antibiotic concentration, the more efficient the process. For instance, the antibiotic concentration can range from two milligrams per liter to ten thousand milligrams per liter. However, this range is not intended to exclude solutions or broths which have been prepared to contain higher concentrations of the antibiotic thienamycin.

One such procedure comprises extracting thienamycin with a strongly acidic liquid cationexchange system at acidic pH, from 2.5-4.5, (forward extraction), separating the phases and then contacting the organic phase with an aqueous back extractant, separating the phases followed by contacting the latter aqueous phase with a strongly basic liquid anion-exchange system at alkaline pH, from 8.0-11.0, (forward extraction), separating the phases and then contacting the organic phase with another aquous back extractant and then separating the phases. The latter aquous solution so obtained can be further purified by the following processes: desalting and chromatography on a polymeric adsorbent, chromatography on an anion-exchange resin of the polystyrene quarternary ammonium type or chromatoraphy on a cation-exchange resin with a buffer or water; and gel filtration and chromatography on an adsorbing resin.

The process of the invention can be carried out using either of the liquid ion exchangers without using the other. Thus, one may utilize the liquid anion exchanger to the exclusion of the liquid cation exchanger, or the liquid cation exchanger can be utilized to the exclusion of the liquid anion exchanger.

However, if both liquid ion exchangers are used in sequence, the sequence in which the liquid ion exchangers are utilized is not critical. Thus, one may use the liquid cation exchanger followed by the liquid anion exchanger or the liquid anion exchanger followed by the liquid cation exchanger.

The extractor system used in this invention can be any of those well known for the separation of liquids having different densities. It will be appreciated that different centrifuges with varying sizes and shapes will have to be adjusted for optimum results.

EXAMPLE 1 A tube of lyophilized culture containing a thienamycin-producing Streptomyces cattleya is opened aseptically and the contents suspended in a 250-ml. baffled Erlenmeyer flask containing 50 ml. of sterile Medium B having the following composition: Medium B Autolyzed yeast type pH 10 g./l.

Dextrose 10 g./l.

Mg S047 H20 50 mg./l.

KH2PO4 0.182 g./l.

Na2HPO4 0.19 g./l.

pH 6.5 before sterilization The inoculated flask is shaken at 28"C on a 150 rpm rotary shaker for 24 hours. Three ten-ml. portions of the Medium B stage 24-hour broth are removed aseptically. Each 10-ml.

portion is mixed immediately with 500 ml. of Medium B contained in three 2-liter baffled Erlenmeyer flasks. These seed flasks are shaken at 28"C on a 150 rpm rotary shaker for 24 hours.

Fifteen hundred ml. of the 24-hour Medium B broths contained in the 2-liter baffled Erlenmeyers are used immediately to innoculate a 756-liter stainless steel fermentor containing 467 liters of Medium E having the following composition: Medium E Glycerol 10 gal Pharmamedia 5 g./l.

CaC12-6H20 0.01 g./l.

Distillers Solubles 10 g./l.

CaC03 3 g./l.

Polyglycol 2000 2.5 g./l.

pH 7.3 before sterilization This tank is operated at 28"C. using an agitation rate of 130 rpm and an airflow of 10 cu.ft.

per minute for 48 hours. The pH of the fermentation is monitored at 24-hour intervals and tabulated in the following table.

Age (hrs.) pH 0 6.8 24 6.8 48 6.5 454 of the above 48-hour broth contained in the 756-liter stainless steel fermentor is used immediately to innoculate a 5670-liter stainless steel fermentor containing 4082 liters of Medium G having the following composition: Medium G Cornsteep liquor 15 g.ll.

Glycerol 10 g./l.

Pharmamedia 5 g./l.

CoCl2 6H20 0.01 g./l.

CaC03 3 g./l.

Polyglycol 2000 2.5 g./l.

pH 7.3 before sterilization This tank is operated at 25"C. using an agitation rate of 0.0154 rpm/liter and an airflow of 0.012 cu.ft./liter for 96 to 100 hours. The batch pH is controlled at 6.0-7.0.

The 4082 liters of fermentation broth is filtered using a 30-inch filter press and a filter aid admix to the extent of 4%w/v. 12g of (ethylenedinitrile)-tetraacetic acid sodium salt is added to the filtrate. The filtrate is cooled to 6"C.

The filtered broth, at about 5"C., is mixed continuously with 2.5 normal sulfuric acid to bring the broth pH to 3 using an in-line mixer. The acidified broth pH 3 is then fed at a rate of 60 U.S. gallons per minute (gpm) to a centrifugal extractor where it is contacted with cold (about 5"C.) 10% v/v dinonylnaphthalenesulfonic acid (DNNS) (primarily in the sodium cycle), at pH 2 in n-butanol solution which is being fed to the extractor at 30 gpm. In the extractor, the two solutions are intimately mixed and the cation exchange reaction occurs between the Na+ and H+ ions of the DNNS moiety and the ammonium cation form of thienamycin, resulting in the transfer of thienamycin from the aqueous phase to the solvent phase. The two phases are then efficiently separated by a Podbielniak Model D-36 operating at 200 rpm, generating centrifugal forces up to 2000 G in the extractor. The thienamycin containing solvent phase, the rich DNNS/solvent stream, is then pumped at 30 gpm to a second extractor where it is contacted with an aqueous buffer, 6%v/v pyridine containing 5 liter Na phosphate dibasic, for the back extraction. The back extractant is fed at the rate of 30 gpm to the extractor being used for the back extraction.

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/nbutanol phase into the aqueous buffer using the second extractor. Thus, the overall thienamycin recovery is about 93% from broth into aquous buffer via the liquid cation exchange process.

The aqueous back extractant from the liquid cation exchange process is now mixed with 2.5 normal sodium hydroxide to bring the pH to 11 using an in-line mixer. This aquous stream, at about 5"C and pH 11, is fed at 30 gpm to athird extractor when it is intimately contacted with a liquid anion exchanger 30% v/v "Aliquat 336", tricaprylyl methyl ammonium acetate (acetate cycle), in n-butanol which is being fed at 30 gpm ("Aliquat" is a trade mark). The anion-exchange reaction occurs between the negative acetate ion of the "Aliquat 336" moiety and the carboxylate anion form of thienamycin resulting in the transfer of thienamy cin from the aqueous phase to the solvent phase. The two phases are then efficiently separated by the centrifugal forces operative in the extractor. The thienamycin-containing solvent phase, the rich Aliquat/solvent stream, is then pumped to a fourth extractor where it is contacted with an aqueous buffer, 0.40 molar potassium acetate (pH 5.0), and again, via anion exchange reactions, the thienamycin is transferred, this time from the solvent phase to the aqueous buffer phase. The spent solvent phase, containing the "Aliquat 336" is then continuously regenerated to the acetate cycle using conventional liquid extraction columns.

By this procedure described, about 80 to 85% of the thienamycin is transferred from the feed aqueous stream to the back extractant buffer vie the liquid anion exchange process.

Substantial purification of the thienamycin results. Purity refers to the ratio of thienamycin titer to the total dissolved solids titer. In the Example, the purity was increased 30 fold.

If desired, the thienamycin-containing extract from the liquid anion exchange process is pH adjusted to 7-7.2 and concentrated to a volume of 20 gal. which is then applied to a 100-gal.

Amberlite XAD-2 column at a rate of 2.5 gal/min. The resin is eluted with deionized water at 5 gal/min and a 150-gallon rich cut is collected and concentrated to 2.5 liters which is then applied to 40 liters of Dowex 50 x 2 (Na+ cycle) resin at a rate of 600ml/ min. The resin is then elated at 600 ml./min. with deionized water and a 17-gallon rich cut collected and concentrated to 0.06 U.S. gal. which is applied to a 30-liter bed of Bio-Gel P-2 (200-400 mesh) previously equilibrated with 0.1M 2,6-lutidine acetate pH 7.0 buffer. The gel is then developed with the same buffer. The rich cut is concentrated to 0.05 U.S. gallon and applied to 4 liters of Amberlite XAD-2 resin. The rich eluate is concentrated and the concentrate freeze-dried, yielding thienamycin, about 90% pure.

WHAT WE CLAIM IS: 1. A method of recovering thienamycin from fermentation broths or solutions containing it that comprises transferring the antibiotic into a solution of a water-insoluble liquid ion exchanger in an organic solvent (forward extraction) and then bringing the liquid ionexchange system into contact with an aqueous buffer solution to transfer the antibiotic into the aqueous buffer phase (back extraction), in which the ion exchanger is (a) a liquid cation exchanger; (b) a liquid anion exchanger; (c) a liquid cation exchanger followed by a liquid anion exchanger; or (d) a liquid anion exchanger followed by a liquid cation exchanger.

2. A method as claimed in claim 1, in which the liquid cation exchanger is a dinonylnaphthalenesulfonic acid or a salt of it.

3. A method as claimed in claim 1, in which the liquid anion exchanger is a tricaprylyl methyl ammonium acetate, tricaprylyl methyl ammonium propionate, tricaprylyl methyl ammonium phosphate or tricaprylyl methyl ammonium sulfate.

4. A method as claimed in any preceding claim, in which the solvent has a moderately high dielectric constant.

5. A method as claimed in claim 4, in which the solvent is a straight-chain or branched chain C4-1o alcohol.

6. A method as claimed in claim 4, in which the solvent is a straight-chain or branchedchain C4-s ketone.

7. A method as claimed in claim 4, in which the solvent is a straight-chain or branched chain C4.10 ester.

8. A method as claimed in claim 1 substantially as hereinbefore described in the Example.

9. Thienamycin when recovered by a method as claimed in any one of the preceding

Claims (1)

1. claims.