GB1592807A - Production of an antibiotic - Google Patents

Abstract

The novel compound of the formula <IMAGE> and its pharmaceutically acceptable salts are prepared by culturing an appropriate strain of Streptomyces flavogriseus in aqueous nutrient medium which contains assimilable sources of carbon, nitrogen and inorganic salts, under submerse, aerobic conditions and recovering the antibiotic formed. The novel antibiotic is active against various gram-positive and -negative bacteria and is a potent inhibitor of bacterial beta -lactamases. It can be used in human and veterinary medicine and is also suitable as an additive to animal feed owing to preservation of feedstuffs and foodstuffs, and as a disinfectant.

Description

(54) PRODUCTION OF AN ANTIBIOTIC (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 discovery of the remarkable antibiotic properties of penicillin stimulated great interest in this field which has resulted in the finding of many other valuable antibiotic substances. In general, the antibacterial activity of each of these antibiotics does not include certain clinically important pathogenic bacteria. For example, some are principally active against only gram-positive types of bacteria. Acquired resistance over the course of widespread use of existing antibiotics in the treatment of bacterial infection has caused a serious resistance problem to arise.

Accordingly, the deficiencies of the known antibiotics have stimulated further research to find other antibiotics which will be active against a wider range of pathogens as well as resistant strains of particular microorganisms.

This invention is concerned with the production of an antibiotic agent herein designated 890as in dilute form, as crude concentrate and in pure form. In accordance with the present invention, the said antibiotic 890at is produced by cultivating a 890Ag-producing strain of Streptomyces flavorgriseus in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions and recovering said antibiotic.

Based upon extensive taxonomic studies, the strain of microorganism used in the process of the present invention was identified as belonging to the species Streptomyces flavogriseus and has been designated MA-4638 in the culture collection of Merck & Cd. Inc,., Rahway, New Jersey. A culture thereof has been placed on permanent deposit without restrictions as to availability with the culture collection of the Northern Regional Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. Peoria, I11., and is available to the public under accession No. NRRL 11,020.

Streptomyces flavogriseus MA-4638 produces antibiotic 890at which is isolated in substantially pure form from the fermentation broth.

The morphological and cultural characteristics of Streptomyces flavogriseus MA-4638 are set forth in the following table.

Morphology Sporophores are branching, straight to flexuous chains of spores, forming tufts. Chains are more than 10 spores in length. Spores are spherical to oval-0.9 uxl.2 L (970x).

Culteral Characteristics Oatmeal agar (ISP Medium 3) Vegetative growth-Reverse-yellow-tan edged with brown, wrinkled; Aerial mycelium-Light gray edged with medium gray Soluble pigment-None.

Czapek Dox agar (sucrose nitrate agar) Vegetative Growth-Reverse-brown edged with dark brown; Aerial mycelium-Medium gray, velvety; Soluble Pigment-Slight browning of medium.

Egg albumin agar Vegetative growth-Reverse-yellow-tan edged with brown; Aerial mycelium-Medium gray mixed with yellowish gray (2dc) and Grayed yellow (2db): soluble pigment-Light yellowish tan.

Glyceros asparagine agar Vegetative growth-Reverse-brown; Aerial mycelium-Velvety, light gray with yellowish tone (2dc); Soluble pigment-Light tan.

Inorganic salts-starch agar (ISP Medium 4) Vegetative growth-Reverse-greenish-yellowish-tan; Aerial mycelium-velvety, medium gray with yellow tone (3fe); Soluble pigment-Very light tan.

Yeast extract-dextrose+salts agar Vegetative growth-Reverse-dark brown; Aerial mycelium-Dark gray mixed with a lighter gray; Soluble pigment-None.

Yeast extract-malt extract agar (ISP Medium 2) Vegetative growth-Reverse-brown: Aerial mycelium-Velvety, dark gray edged with a lighter gray: Soluble pigment-None.

Skim milk agar Vegetative growth-Tan; Aerial mycelium-Spanse, whitish; Soluble pigment-Slight browning of medium; Hydrolysis of casein-Good.

Litmus milk Vegetative growth-Moderate growth ring, tan; Aerial mycelium-None; Color-Purple; coagulation and/or peptonization-Complete peptonization: becoming alkaline.

Skim milk Vegetative growth-Moderate growth ring, tan; Aerial mycelium-None; Soluble pigment-Light brown; Coagulation and/or peptonization-Complete peptonization; becoming alkaline.

Nutrient tyrosine agar Vegetative growth-Reverse-dark brown; Aerial mycelium-Dark gray edged with grayish white; Soluble pigment-Slight browning of medium; Decomposition of tyrosine-Positive.

Peptone-iron-yeast extract agar Vegetative growth-Tan; Aerial mycelium-whitish, moderate; Soluble pigment-None; Melanin-None; HvS production-Negative.

Nutrient agar Vegetative growth-Reverse-light grayish brown; Aerial mycelium-Light gray edged with dark gray; Soluble pigment-None.

Nutrient starch agar Vegetative growth-Tan edged with gray; Aerial mycelium Medium gray; Soluble pigment-None; Hydrolysis of starch-Good.

Nutrient gelatin agar Vegetative growth-Tan edged with gray; Aerial mycelium-Grayish-white; Soluble pigment-None; Liquefaction of gelatin-Good.

Potato plug Vegetative growth-Tan; Aerial mycelium-Medium to dark gray; Soluble pigment-None.

Loeffler's Blood serum Vegetative growth-Cream-colored; Aerial mycelium-None; Soluble pigment-None; Liquefaction--None.

Gelatin stabs Vegetative growth-Tan; Aerial mycelium-None; Soluble pigment-None; Liquefaction of gelatin-Complete.

All of the readings reported above were taken after three weeks incubation at 28"C. unless noted otherwise. the pH of the media used in these studies was approximately neutral, namely, pH 6.8-7.2. The color designations used in the description are in accordance with the definitions of the Color Harmony Manual, 4th Edition (1958), Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus MA-4638 was also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown on basal synthetic medium (Pridham and Gottlieb) containing 1 /,, of the carbohydrate at 280C. for three weeks. The pH of the media employed in the study was approximately neutral (6.8-7.2). Table I shows the utilization of these carbohydrate sources by Streptomyces tlavogriseus MA-4638; +indicating growth, +poor or questionable growth, and no growth as compared to negative control (no carbon source).

TABLE I Glucose + Maltose + Arabinose + Mannitol + Cellulose - Mannose + Fructose + Raffinose Inositol - Rhamnose + Lactose + Sucrose + Xylose + The amount of growth with change in temperature and the oxygen requirement by the microorganism is as follows: Temperature range (Yeast extract-dextrose+salts agar); 28"C.--Good vegetative and aerial growth 37"C.--Good vegetative growth; no aerial hyphae 500C.-No growth Oxygen requirement (Stab culture in yeast extract-dextrose+salts agar); Aerobic.

The present invention is not limited to the organism, Streptomyces,flavogriseus NRRL-11020 or to organisms fully answering the above growth and microscopic characteristics which are given for illustrative purposes. It is desired and intended to include the use of mutants produced from the described organism by various means, such as X-radiation, ultraviolet radiation, nitrogen mustard and phage exposure.

890A9 is produced during the aerobic fermentation, under controlled conditions, of suitable aqueous nutrient media inoculated with a strain of the organism, Streptomyces flavogriseus. Aqueous media, such as those employed for the production of other antibiotics, are suitable for producing 890at. Such media contain sources of carbon, nitrogen and inorganic salts assimilable by the microorganism.

In general, carbohydrates such as sugars, for example, dextrose, glucose, fructose, maltose, sucrose, xylose and mannitol and starches such as dextrin or such as grains, for example, oats, rye, cornstarch and corn meal can be used either alone or in combination as sources of assimilable carbon in the nutrient medium.

The amount of carbohydrate usually varies between 1% and 6% by weight of the medium. These carbon sources can be used individually, or several such carbon sources may be combined in the medium. In general, many proteinaceous materials may be used as nitrogen sources in the fermentation process. Suitable nitrogen sources include, for example, yeast hydrolysates, primary yeast, soybean meal, cottonseed flour, hydrolysates of casein, corn steep liquor and distiller's solubles, the preferred source being distiller's solubles. The sources of nitrogen, either alone or in combination, are used in amounts ranging from 0.2% to 6% by weight of the aqueous medium.

Among the nutrient inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding ions such as sodium, potassium, ammonium, calcium, magnesium, phosphate, sulfate, chloride and carbonate. Also included are trace metals such as cobalt, manganese and iron.

The media described in the Examples are merely illustrative of the wide variety of media which may be employed, and are not intended to be limitative.

The fermentation is carried out at temperatures ranging from 20"C. to 370 C.; however, for optimum results it is preferable to conduct the fermentation at temPeratures of from 23"C. to 280C. The initial pH of the nutrient media suitable for growing strains of the Streptomyces flavogriseus culture and producing antibiotic 890at can vary from 6.0 to 8.0.

Although antibiotic 890at can be produced by both surface and submerged cultures, it is preferred to carry out the fermentation in the submerged state.

A small-scale fermentation of the antibiotic is conveniently carried out by inoculating a suitable nutrient medium with the antibiotic-producing culture and, after transfer to a production medium, permitting the fermentation to proceed at a constant temperature of about 24"C. on a shaker for several days.

The fermentation is initiated in a sterilized flask of nutrient medium via one or more stages of seed development. The nutrient medium for the seed stage may be any suitable combination of carbon and nitrogen sources. The seed flask is shaken in a constant temperature chamber at about 28"C. for one day, or until growth is satisfactory, and some of the resulting growth is used to inoculate either a second stage seed or the production medium. Intermediate stage seed flasks, when used, are developed in essentially the same manner; that is, part of the contents of the flask from the last seed stage are used to inoculate the production medium. The inoculated flasks are shaken at a constant temperature for several days, and at the end of the incubation period the contents of the flask are centrifuged or filtered.

For large-scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium.

According to this method, the nutrient medium is made up in the tank and sterilized by heating at temperatures of up to 1200C. Upon cooling, the sterilized medium is inoculated with a previously grown seed of the producing culture, and the fermentation is permitted to proceed for a period of time as, for example, from 1 to 6 days while agitating and/or aerating the nutrient medium and maintaining the temperature at 22 to 260C. This method of producing antibiotic 890at is particularly suited for the preparation of large quantities of the antibiotic.

Physical and Chemical Properties of Antibiotic 890at Antibiotic 890at is an acidic substance which migrates toward the positive pole on electrophoresis at neutral pH. At the gradient of 50 volts/cm. in 0.03M potassium phosphate buffer, pH 7.1, the antibiotic moves 8.0 cm. in 30 minutes, compared with movement of 4.0 cm. for 890A,. The disodium salt is a white or slightly yellow powder as lyophilized from aqueous solution. Under acidic conditions in aqueous solution, the antibiotic is unstable in the free acid form.

Therefore, the antibiotic is usually found in a combined form as a salt or other derivative which is more stable.

The disodium salt of antibiotic 890at has absorption maxima at 308 nm and 228 nm, and a minimum at 262 nm at neutral pH in water. For the most highly purified preparation, the A308/A260 ratio is 2.05, and the A308/A220 ratio is 1.17 and the A30,,/A228 ratio is 1.05. More than 93% of the absorbance at 308 nm may be extinguished by reaction with hydroxylamine at neutral pH. Upon reaction with hydroxylamine, the absorbance at 260 nm increases, and the ratio of the increase at 260 nm to the decrease at 308 nm is approximately 0.30. The reaction with hydroxylamine, as followed by A308 decrease under the conditions described in the section "Hydroxylamine Reaction" is apparently first order, with a half-life at room temperature of from 25 seconds to 50 seconds.

When measured against a standard of antibiotic 890A1, the antibiotic 890A9 has 82 units per HAEA308 unit. HAEA308 is described hereinafter under the section "Hydroxylamine Reaction".

Table II lists the 100 MHz-nuclear magnetic resonance spectral signal of 890A9 in D2O at 10 C. Chemical shifts are given in ppm relative to HOD at 4.70 a at 320C., coupling constants are given in Hertz.

TABLE II CH3CH 1.55 (3H, d, 6Hz) CH3CO 2.12(3H, s) C6-H 3.91 (1H, d, d; J,~,=5.5 Hz; J6~8=9Hz) C5-H 4.36 (IH, m) C8-H 5 (partially covered by HOD line) C1 H2 3.14(1H, d, d; 18.2+9.8 Hz) -ZCH=CH-N 6.10(1H, d, 13.9 Hz) and 7.19 (IH, d, 13.9 Hz) The mass spectra of TMSi-890Ag is characterized by fragments as shown in Table III.

TABLE III m/e 84 227 298/9 339 340 366 456 Antibiotic 890A9 has a molecular structure as follows:

Antibiotic 890A9 is further characterized by the following antibiotic spectrum profiles.

The test to determine the antibiotic spectrum profiles of antibiotic 890Ag is carried out by application of a 0.015 ml. droplet of a 20 Mg./ml. aqueous solution of the antibiotic on the surface of a 100x 15 mm. petri plate containing 5 ml. of seeded nutrient agar plus 0.2 / yeast extract which is incubated at 250C. The results, expressed in terms of the diameter in millimeters of the zone of inhibition, are set forth in Table IV.

TABLE IV Inhib. Zone Organism Diam., mm Bacillus sp. MB No. 633 38 Proteus vulgaris MB No. 1012 26 Pseudomonas aeruginosa MB No. 979 0 Serratia marcescens ATCC 890 21 Staphylococcus aureus ATCC 6538 P 30 Bacillus subtilis ATCC 6633 38 Sarcina lutea ATCC 9341 30 Staphylococcus aureus MB No. 698 20 Streptococcus,faecalis MB No. 753 0 Alcaligenes faecalis ATCC 213 29 Brucella bronchiseptica ATCC 4617 16 Salmonella gallinarum MB No. 1287 34 Vibrio percolans ATCC 8461 32 Xanthomonas vesicatoria MB No. 815 28 Proteus vulgaris ATCC 21100 35 TABLE IV (cont.) Inhib. Zone Inhib. Zone Organism Diam., mm Escherichia coli MB No. 1418 30 Pseudomonas stutzeri ATCC 11607 10 Klebsiella pneumoniae MB No. 1264 24 Aerobacter aerogenes MB No. 835 25 Er win ia atroseptica ATCC 4446 30 Pseudomonas aeruginosa M B No. 2824 0 Corynebacterium pseudodiph. ATCC 9742 19 Escherichia coli ATCC 9637 26 Streptococcus ,faecium M B No. 2820 10 Streptococcus agalactiae MB No. 2875 29 Vibrio percolans MB No. 2566 (res. ceph C) 21 Proteus vulgans MB No. 2112 (episome) 34 Proteus mirabilis MB No. 3126 27 Vibrio percolans ATCC 8461+2xl05 u/ml penicillinase 19 Vibrio percolans ATCC 8461+ -lactamase from Enterobacter clacae MB 2646 32 Antibiotic 890at is active against various gram-positive and gram-negative bacteria and is a potent inhibitor of bacterial p-lactamases, and may find utility in human and veterinary medicine. 890at may be used alone or in combination with other antibacterial drugs for treating infections caused by gram-positive or gramnegative bacteria, for example, against Staphylococcus aureus. Proteus mirabilis.

Escherichia coli, Klebsiella pneumoniae and Salmonella schottmuelleri.

The compound of this invention may also be used in conjunction with p-lactam antibiotics susceptible to p-lactamases, to potentiate the action of such p-lactam antibiotics by inhibiting the lactamase activity and thus prolonging the life time of the antibiotics.

Thus, a combination of antibiotic 890A > , with lactamase-sensitive p-lactam antibiotic will be more efficacious for the treatment of infection with p-lactamase- producing bacteria than would the same quantity of p-lactamase-sensitive antibiotic alone.

Antibiotic 890at and its non-toxic pharmaceutically acceptable salts may be incorporated in antibacterial amounts in a non-toxic pharmaceutical carrier. In addition they may be utilized as an additive to animal feeding stuffs, for preserving foodstuffs and as disinfectants. It may be included in aqueous compositions in amounts ranging from 0.1 to 100 parts of antibiotic per million parts of solution or preferably in concentrations ranging from I to 10 parts of antibiotic per million parts of solution in order to destroy and inhibit the growth of harmful bacteria on medical and dental equipment and as bactericides in industrial applications.

Antibiotic 890at may be used in pharmaceutical preparations as the sole active ingredient or in combination with one or more other antibiotics or with one or more pharmacologically active substances.

The antibiotic may be administered orally, topically, intravenously or intramuscularly. The methods employed for administration may be any that are well known in the art or any to which the antibiotic herein described may be adapted.

Also, in addition to a carrier, the compositions may include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricators, suspending agents, viscosity agents or flavoring agents that are well known and commonly used.

In veterinary medicine, such as in the treatment of cows, sheep and pigs, the composition may, for example, be formulated as an intramammary preparation in either long acting or quick-release bases. The antibiotic may also be used in treatment of chickens.

The dosage to be administered depends to a large extent upon the condition of the subject being treated, the weight of the host and the type of infection, the route and frequence of administration, the parenteral route being preferred for generalized infections and the oral route for intestinal infections.

In the treatment of bacterial infections in man, the compound of this invention may be co-administered with a p-lactamase-sensitive antibiotic orally or parenterally, in accordance with conventional procedures for antibiotic administration, the antibiotic 890at being given, in an amount of from 2 to 600 mg./kg./day and preferably 5 to 100 mg./kg./day in preferably divided dosage, e.g.

three or four times a day. It may be administered in dosage units containing, for example, 100, 330, 400 or 1000 mg. of active ingredients with suitable physiologically acceptable carriers or excipients. The dosage units are in the form of liquid preparations such as solutions or suspensions or as solids in tablets or capsules. It will, of course, be understood that the optimum dose in any given instance will depend upon the type and severity of infection to be treated, and that pediatric doses will be smaller all of such adjustments being within the skill of the practitioner in the field.

Included in this invention are the non-toxic, pharmaceutically acceptable salts of 890at. For example, the pharmacologically acceptable salts formed with inorganic and organic bases; which include, for example, metal salts derived from alkali metal or alkaline earth metal hydroxides, carbonates, or bicarbonates, such as those derived from sodium, potassium, ammonium and calcium and salts derived from primary, secondary or tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-loweralkanolamines, lower alkylenediamines, N,N-diaralkyl lower alkylenediamines, aralkylamines, amino substituted lower alkanols, N,N-di-lower alkylamino substituted lower alkanols, amino-polyamino and guanidino-substituted lower alkanoic acids and nitrogencontaining heterocyclic amines. Representative examples include salts derived from sodium hydroxide, ammonium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, trimethylamine, triethylamine, piperidine, N-ethylpiperidine, morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl- 1 -propanol, theophylline and Nmethylglucamine.

The salts of the compounds of the present invention may be isolated direct!y from fermentation media by the use of appropriate eluents during the ion-exchange chromatography or prepared by conventional methods. For example, the di-salts such as disodium salt may be obtained by treating two equivalents of sodium hydroxide with one mole of the product (I) in a suitable solvent. Also mixed salts with monovalent cations may be prepared by combining one mole of a monovalent base with one mole of the product (I) plus one equivalent of another base.

Alternatively, monobasic salts may be obtained by treating one equivalent of a base having a monovalent cation with one mole of the product (I). Also, salts may be formed by treating one mole of the product with one mole of a base having a divalent cation. The salts of this invention are pharmacologically acceptable nontoxic derivatives which can be used as the active ingredient in suitable unit-dosage pharmaceutical forms. Also, they may be combined with other drugs to provide compositions having a broad spectrum of activity.

Fermentation broths containing the antibiotic 890at produced in accordance with the procedures described herein have activities ranging from about 2 to 170 units per ml. when assayed in accordance with the disc-diffusion assay using Vibrio percolans (ATCC 8461). The antibiotic 890at contained in these fermentation broths can be recovered and purified by a number of procedures, and in the description that follows the words "Dowex", "Duolite", "Amberlite", "Sephadex" and "Difco" are trade marks and mesh sizes are U.S. standards. One such procedure comprises adsorbing the antibiotic 890Ag on a strongly basic anion exchange resins are those having a styrene-divinylbenzene matrix, for example the polystyrene nuclear quaternary ammonium resin Dowex 1 x2 (manufactured by Dow Chemical Co., Midland, Michigan), on the chloride cycle. Other representative members of this class of strongly basic exchange resins include the following: Duolite A-40, A42, A-101, A-102 and A-114 (manufactured by Chemical Process Co., Redwood City, California). Amberlite IRA-400, IRA-401 and IRA410. Alternatively, a weakly basic anion exchange resin such as Amberlite IRA-68 may be used. (Amberlite resins are manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania).

The adsorbed antibiotic is readily eluted from the anion exchange resin with salt solutions in 80 /^ (v/v) aqueous methanol. The eluate so obtained can be further purified by other purification procedures. Thus, the eluate can be purified by concentrating it and passing it through a column packed with a polystyrene, nonpolar, hydrophobic crosslinked divinyl benzene polymer such as XAD- 1. 2 and 4, or polyacrylamide resins such as XAD-7 and 8. XAD-2 is preferred, (XAD-I. 2, 4, 7, and 8 are manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania).

A method of obtaining further purified antibiotic 890As is by the use of gel filtration through polyacrylamide gel having a pore size which excludes molecules having a molecular weight greater than 1800, such as Bio-Gel P-2 (manufactured by Bio Rad, Richmond, California). Other gels, such as Sephadex G-l0 may also be employed for desalting.

The preferred procedure by which antibiotic 890at may be obtained in high purity from a broth consisting of centrifugation or filtration of the broth to remove solids; an adsorption and elution of the filtrate from an anion-exchange resin such as Dowex-l x2 in the chloride cycle with 3% NaCI in 80 " (v/v) aqueous methanol, which both concentrates and partially purifies the antibiotics: a passage over a column of suitably prepared XAD-2, which retards the antibiotics and thereby purifies and desalts the Dowex-lx2 eluate. The fractions enriched in 890at are pooled and further purified. Chromatography on a Dowex-l x2 minus 400 mesh resin, with elution by NaCI and/or NH4Cl in 80% aqueous methanol, gives a product free from most UV-absorbing impurities (the NH4Cl is used to provide some buffering capacity in the eluant) and separates 890at from other antibiotics; a desalting on Bio-Gel P-2 of Sephadex G-10 in 50% methanol, removes most of the salt introduced in the Dowexlx2 chromatography.

The remaining impurities may be reduced by an additional cycle of chromatography on Dowex-lx2, minus 400 mesh, with elution by a solution containing sodium chloride and 50 methanol, followed by desalting.

In purification by colum

1 FIGURE 1 2 Outline of purification Process For Antibiotic 890Ao

3 \ A Centrifuged 4 Fermented 1 Broth S Broth contasn- 1. chill 3 ing 890A9 2. centrifuge 1. adsorb to Dowex-lx2(Cl ) 7 ) 3. add EDZk (50-100 mesh) 8 1 / 2. wash with 0.15M NaCi + .OlM 9 - 0- Zris-HC1 + 25M ElrzA in 50% 10 MH 11 3. wash with water 12 4. elute with 38 NaCl + O.OLM 13 , I Tris-HCl, pH 7 + 25pom neutral 14 . EDTA : n 80% methanol 15 IDcwex-lx2 16 (50-100 mesh) 17 80% XMeaH Eluate 18 1. concentrate le , 2. adsorb to XXD-2 20 3. elute with H20 21 22 Eluate 23 ~ 1. concentrate fractions con 24 taining 890 25 2. dilute with < DS 26 3. adsorb to Dowex-lx4(Cl ) 27 (minus 400 mesh) 28 4. elute with 0.26M Naci + .005to 29 / NH Cl + .00005M NH3 in 80% 30 ~~ ~ me hanoL 31 iDcwex-lK4 32 Elate 890at 33 1. dilute with H20 34 2. adsorb to Dowu -lx2(Cl ) 35 (minus 400 mesh) 36 3. elute with 0.3014 NaC1 + 37 0.005M NH Cl + .0001M 38 f NH3 in SOt methanol 38 39 1x2 (-400 mesh) 40 Eluate 41 1. concentrate 42 2. add 1 vol. MS.CH 43 3. adsorb to Bio-Gei P-2 (in 50% *4 biXDH) 45 4. elute with .02at4 NH3 in 50% 46 47 Bic-Gal 2 48 Eluate 49 1. concentrate 50 2. Lyophilize 51 S Assay Procedures for Antibiotic 890at 1. Bioassay An agar plate disc-diffusion method is employed using Vibrio percokms ATCC 8461 as tester organism. A purified sample of antibiotic 890At is used as standard.

Antibiotic 890at is prepared according to the procedure set forth in Example 4.

Plates containing Vibrio percolans ATCC 8461 are prepared as follows: A lyophilized culture of Vibrio percolans ATCC 8461 is suspended in 15 ml. of a sterilized medium containing 8 g./liter of Difco Nutrient Broth and 2 g./liter of yeast extract in distilled water "nutrient broth-yeast extract" (hereinafter designated NBYE). The culture is incubated overnight on a rotary shaker at 28"C.

This culture is used to inoculate the surface of slants containing 1.5 ," agar in NBYE, and the inoculated slants are incubated overnight at 280C., and then stored in a refrigerator.

The refrigerated slants prepared from a single lyophilized culture are used for up to four weeks from their preparation, as follows: A ioop of inoculum from the slant is dispersed in 50 ml. of NBYE contained in a 250 ml. Erlenmeyer flask. The culture is incubated overnight on a rotary shaker at 280C. and then diluted to a density giving 50 /n transmittance at 660 nm. A 33.2 ml. portion of this diluted culture is added to 1 liter of NBYE containing 15 g. of agar and maintained at 460C.

The inoculated agar-containing medium is poured into 100x15 mm. plastic petri dishes, 5 ml. per dish, chilled, and maintained at 2-40C. for up to 5 days before using.

Filter paper discs of one-half inch diameter are dipped into the solution to be assayed, and are placed on the agar. Alternatively, the discs may be loaded by pipetting one-tenth ml. of solution onto a dry disc, and then placing the disc on the agar. The diameter of the zone of inhibition is measured after appropriate incubation (12-24 hours at 250C.). If necessary, dilutions of the solutions to be assayed are made in 0.05M potassium phosphate buffer, pH 7.4 "potassium phosphate buffer" (hereinafter referred to as KPB), or in deionized water.

Calculations of potencies proceed as follows: a slope is determined by measuring the zone diameters of a solution of antibiotic 890As and of a fourfold dilution (in KPB) of this solution. Two discs of each concentration are assayed on a single plate, and the average zone size at each concentration is determined. The slope is equal to one-half of the difference of the average zone sizes. Potencies are then calculated by the formula:

g [D-DS] log A Potency (units/ml.)= 2 slope (Potency of Standard)xDilutionxl0 where D is the average diameter of the zones formed by the unknown, Ds is the average diameter of the standard zones, and "Dilution" is the degree to which the unknown was diluted before assay. If no standard is used, Ds is assumed to be 25 mm. and (Potency of Standard) is taken as I unit/ml., when measured on Vibrio percolans ATCC 8461. Pure 890A7 is defined as having a potency of 250 units per hydroxylamine-extinguishable absorbance unit at 300 nm, when used as a standard.

II. Assay Procedure for Determining "890 Assay Units" A conventional agar plate disc-diffusion method is employed using Vibrio percolans ATCC 8461 as tester organism. Cephaloridine is employed as a standard.

Plates containing Vibrio percolans ATCC 8461 are prepared as follows. A culture of Vibrio percolans ATCC 8461 is incubated in nutrient broth-yeast extract overnight on a rotary shaker at 280C. and then diluted to a density of 60 / transmittance at 660 nm. A 33.2 ml. portion of this diluted culture is added to 1 liter of a medium composed of nutrient agar plus 0.2% yeast extract maintained at 460C. The inoculated agar-containing medium is poured into 100x 15 mm. plastic-petri dishes, 10 ml. per dish, chilled, and maintained at 240C. for up to 5 days before use.

The concentration of cephaloridine which is equivalent to 1 unit/ml. of 890A1 is determined by assay on plates prepared as above, but containing 5 ml. of inoculated medium per plate, as follows, Four concentrations of cephaloridine constitute the standard-3.12, 6.25, 12.5 and 25 mcg per ml. with the 12.5 mcg per ml. as a reference solution, The zone diameters on a 5 ml. plate for the standard are as follows: Conc. (mcg/ml.) Zone Diameter (mm.) 3.12 16.8 6.25 22.3 12.5 25.0 25 29.6 A unit as defined as the amount of antibiotic per ml. producing a 25 mm. zone of inhibition on a 5 ml. plate as described in section I above. Therefore, in this assay a concentration of 12.5 mcg per ml. of cephaloridine is considered equivalent to I unit of 890A1 per ml. Since the slope of the line for cephaloridine is 4.0 calculations of the potency of a sample are made using a slope of 4.0.

III. Hydroxylamine Reaction Antibiotic 890at reacts with hydroxylamine and produces a substance with greatly diminished absorbance at 308 nm. This provides the basis for a quantitative assay of antibiotic 890at.

The solution to be assayed is brought to 0.05M in potassium phosphate, pH 7.4 by adding 1/20th volume of a solution containing 0.8M K2HPO4 and 0.2M KH2PO4.

The one-hundredth volume of 1 M hydroxylamine hydrochloride is added, and the absorbance at 308 nm is measured at intervals of one half to two minutes. The reaction is conducted at room temperature. First-order kinetics are assumed and a half-life is estimated from the absorbance decrease during the first ten minutes.

From this half-life, the time is estimated beyond which no further absorbance decrease should be observed and observations are continued beyond that time. If no further decrease is observed beyond that time, the total absorbance decrease (correcting for dilution effect and absorbance of the hydroxylamine) is taken as the "Hydroxylamine-extinguishable absorbance at 308 nm (HAEA308)". If absorbance decrease is observed beyond that time, the rate of background absorbance decrease is calculated, and the observed decrease at that time is corrected for background decrease, assuming that background decrease is linear with time. The corrected value is then recorded as the HAEA308.

The number of HAEA308 units is equal to the HAEA308 multiplied by the volume in ml.

The examples which follow illustrate the methods by which the products of this invention may be obtained.

EXAMPLE 1 A slant culture containing MA-4638 is used to inoculate a 250-ml. baffled Erlenmayer flask containing 50 ml. of Medium A.

Medium A Dextrose 10.0 g.

Yeast Autolysate (Ardamine*) 10.0 g MgSO4. 7H2O 0.05 g.

Phosphate Buffer** 2.0 ml.

Distilled Water 1000 ml.

pH 6.5 *Ardamine: Yeast Products, Inc.

**Phosphate buffer solution: KH2PO4 91.0 g.

Na2HPO4 95.0 g.

Distilled Water 1000 ml.

This flask is shaken at 280C. on a 220 rpm shaker, 2-inch throw for two days when growth is satisfactory. After two days, this seed is used to inoculate three 250-ml.

Erlenmeyer flasks containing 40 ml. Medium B using 2 ml. per flask (5 /n).

Medium B Tomato Paste 20.0 g.

Whole Oats (ground) 20.0 g.

Distilled Water 1000 ml.

pH: adjust to 7.0 using NaOH These production flasks are also shaken at 280C. on a 220 rpm shaker for up to four days with assays run during the fermentation. At three days age, an aliquot of the supernatant from centrifuged broth is submitted for classification and identification studies. Using 1/4 inch assay discs on standard assay plates, this broth gives a 22 mm. zone of inhibition against Proteus vulgaris and a 15 mm. zone against Salmonella gallinarum. Bioautography of an electrophoretogram of the centrifuged broth shows two components. The faster moving smaller spot contains 890at.

EXAMPLE 2 Fifteen frozen vials containing 1 ml. of MA-4638 culture broth are slowly thawed and the contents aseptically transferred to fifteen 250-ml. triple-baffled Erlenmeyer flasks containing 50 ml. of C seed medium. The flasks are stoppered with cotton.

C Medium Autolyzed Yeast (Ardamine*) 10.0 g.

Glucose 10.0 g.

MgSO4.7H2O Hz 7Hz 0.05 g.

Phosphate Buffer** 2.0 ml.

Distilled Water 1000 ml.

pH adjusted to 6.5 with NaOH before autoclaving *Ardamine: Yeast Products, Inc.

**Phosphate buffer solution: KH2PO4 91.0 g.

Na2H PO4 95.0 g.

Distilled Water 1000 ml.

The seed flasks are shaken for 2024 hours at 280C.ll0C. on a 210 rpm gyrotory shaker, 2-inch throw. Broth from the seed flasks is used to inoculate production flasks.

Different production flasks containing D production medium are used: six 2liter unbaffled shake flasks containing 150 ml. medium per flask, cotton closure; eight 2-liter triple-baffled shake flasks containing 350 ml. of medium per flask, gauze closure; and 250 of 250 ml. unbaffled shake flasks containing 40 ml. medium per flask, cotton closure. the levels of inocula used are: 5 ml. per 150 ml. medium: 10 ml. per 350 ml. medium; and 1.5 ml. per 40 ml, medium.

D Medium Dextrin (CPC Modified Starch) 40.0 g.

Distiller's Solubles 7.0 g.

Yeast Extract 5.0 g.

CoCI2 6H2O 50.0 mg.

Distilled Water 1000 ml.

pH adjusted to 7.3 with NaOH before autoclaving After inoculation, the production flasks are incubated at 250C.ll0C. with shaking on a 220 rpm gyrotory shaker, 2-inch throw for three days. The flasks are harvested, and the contents pooled.

The broth is centrifuged in 200-ml. portions at 11,000 RPM for 15 minutes. To the combined supernatants, totalling 9 liters, are added 2 ml. of 0. I M EDTA, and the supernatant is applied to a column (8.2x20 cm.) of Dowex-lx2(C1), 50100 mesh, which has been previously washed with 5 liters of 0. I M HC1+30 g./l. NaCI in 80 /, (v/v) aqueous methanol, followed by 5 liters of deionized water. The rate of application is from 10 ml./minute to 50 ml./minute. After application of sample, the column is washed with 1 liter of deionized water followed by 9 liters of 0.15 M Nail+0.01 M Tris-HC1 buffer, pH 7.0+25 Mm neutral EDTA in 50% aqueous methanol. The antibiotic 890Ag is then eluted with 9 liters of 30 g./l. NaCl+0.0l M Tris-HC1 buffer, pH 7.0+25 pM neutral EDTA in 80% aqueous methanol, at a flow rate of 40 ml./minute. Fractions of from 200 to 900 ml. are collected.

Antibiotic activity by ATCC 8461 assay appears in all fractions, 1 to 19, with a broad maximum at fractions 6 to 11, extending from 1290 to 3550 ml. of eluted volume, Fractions 6 to 14 are combined and concentrated under reduced pressure to 190 ml., and the pH is brought from 7.6 to 6.5 by addition of 0.4 ml. of 12 M HCI.

The concentrate is applied to a column (4.95x36 cm.) of XAD-2 which has been previously washed with 4 liters each of 60% aqueous acetone, deionized water, and 50 g./l. of NaCI in deionized water. After application of sample, the column is washed three times with 10 ml. of deionized water, and is eluted with deionized water at 15 ml./minute. Fractions of from 100 to 500 ml. are collected.

This column is washed, and the chromatography conducted, at room temperature, and the eluted fractions are immediately chilled in ice water.

Antibiotic activity by ATCC 8461 assay appears in fractions 4 to 12, extending from 450 to 2345 ml. of eluted volume. Fractions 5 to 8, extending from 600 to 1245 ml. of eluted volume, had the highest ratios of HAEA304/A220, and are consequently combined for further purification. The pooled fractions have a total of 607 HAEA304 units.

The combined fractions 5 to 8 are concentrated under reduced pressure to 40 ml. and the concentrate is diluted with 160 ml. of methanol. This solution is adjusted to pH 7.5 with 1 M NaOH, and is applied on a column 2.2x40 cm. of Dowex-lx4(C1-), minus 400 mesh, which has been previously washed with 2 liters of 0. I M He1+30 g./l. NaCI in 800j" (v/v) aqueous methanol, followed by 1 liter of 80% methanol. the rate of application of sample is 2 ml./minute. After application, the column is washed with 50 ml. of 80% methanol, and is eluted with 0.26 M NaCl+0.0l M NH4Cl+0.0002 M NH3 in 80% methanol, at 2 ml./minute. Fractions of 10 ml. are collected.

Bioactivity by ATCC 8461 assay appears in fractions 87 to 280. The main peak of antibiotic 890As appears in fractions 231 to 275, the maximum activity being at fraction 250. Fractions 231 to 275 are combined, comprising the 890at Dowex pool.

The 890at Dowex pool (combined fractions 231 to 275) is diluted with 1600 ml.

of deionized water and is applied on a column (2.2x42 cm.) of Dowex-lx2(C1-) minus 400 mesh at 2 ml./minute. After application, the column is washed with 50 ml. of 50% (v/v) methanol, and is eluted with 3.4 liters 0.30 M Nay1+0.010 M NH4Cl+0.0002 M NH3 in 50% methanol, followed by two liters of 0.30 M Nail+0.01 M NH4C1+0.0002 M NH3 in 60% methanol, at 2 ml./minute. Fractions of 9.5 ml. are collected.

The main peak of absorbance at 305 nm appears in fractions 385 to 440, with a maximum at fraction 410. Fractions 400 to 425 have the highest A305/A260 ratios, and are pooled for further processing. The combined fractions contain 160 A305 units, of which 147 are hydroxylamine-extinguishable.

The combined fractions are concentrated to 5.5 ml. under reduced pressure.

The pH is adjusted to 7.1 by addition of 30 ul. of I M NaOH, and 5.5 ml. of methanol are added. The sample is then applied on a column (2.2x69 cm.) of Sephadex G-10 which has been previously washed with two liters of 0.05 mM NH3 in 50 /n (v/v) methanol. After the sample is applied, the column is eluted with 0.05 mM NH3 in 50 /" methanol, at 1 ml./minute, and fractions of 2.95 ml. are collected.

The main peak of absorbance at 307 nm appears in fractions 69 to 85, with a maximum at fractions 77 and 78. Conductivity measurements on individual fractions show that the main peak of absorbance at 307 eluted somewhat after the peak of salt. Fractions 74 to 82 are combined, containing 68 A307 units, with a ratio A307/A260 of 1.80.

The combined fractions are concentrated to 3 ml. under reduced pressure, and the concentrate is applied on a column (3.4x45 cm.) of Dowex-SOx2 (Na+), 200- 400 mesh, which has been previously washed with 2 liters of 10-5 M NaOH in deionized water, followed by 100 ml. of deionized water. After application of sample, the column is rinsed twice with 2 ml. of deionized water, and then eluted with deionized water at 4 ml./minute. Fractions of 3.95 ml. are collected.

The main peak of absorbance at 307 nm appears in fractions 38 to 48, with a maximum at fractions 42 and 43. Fractions 42, 43, and 44 have the highest A307/A220 ratio, and are pooled for lyophilization. The pooled fractions are adjusted from pH 5.8 to pH 7.0 by addition of 2.2 ul. of 1 M NaOH, and 2.5 ml. are removed. The remaining 9.4 ml. are concentrated under reduced pressure to 1 ml., and 10 ml.

D2O are added. The concentration to 1 ml. and addition of 10 ml. D2O are repeated, and a final concentration is performed. The 1 ml. of concentrate is frozen and lyophilized, giving 20 mg. of solids of which approximately 18 mg. are NaCI as estimated by conductivity.

EXAMPLE 3 Two frozen vials each containing 1 ml. of MA-4638 culture broth are slowly thawed, and the contents are aseptically transferred to two 250-ml. triple-baffled Erlenmeyer flasks, each containing 50 ml. of C seed medium. The flasks are stoppered with cotton.

C Medium Autolyzed Yeast (Ardamine*) 10.0 g.

Glucose 10.0 g.

MgSO4. 7H2O 0.05 g.

Phosphate Buffer** 2.0 ml.

Distilled Water 1000 ml.

pH adjusted to 6.5 with NaOH before autoclaving *Ardamine: Yeast Products, Inc.

**Phosphate buffer solution: KH2PO4 91.0 g.

Na2HPO4 95.0 g.

Distilled Water 1000 ml.

The seed flasks are shaken for 2024 hours at 280C.+10C. on a 210 rpm gyrotory shaker, 2-inch throw. The contents are pooled and used to inoculate production flasks.

Ten 2-liter baffled shake-flasks, each containing 300 ml. of D production medium, are inoculated with 10 ml. per flask of the broth from the seed flask. The production flasks are covered with gauze closures.

D Medium Dextrin (CPC Modified Starch) 40.0 g.

Distiller's Solubles 7.0 g.

Yeast Extract 5.0 g.

CoCI2 6H2O 50.0 mg.

Distilled Water 1000 ml.

pH adjusted to 7.3 with NaOH before autoclaving After inoculation, the production flasks are incubated at 240C+1"C. with shaking on a 210 rpm gyrotory shaker, 2-inch throw, for three days. The flasks are harvested, contents pooled and the broth is assayed for activity.

Harvest Age (hours) 72 pH 6.5 890 Assay (units/ml.) 30.45 Two liters of whole broth from the above KR production flasks are centrifuged in 200-ml. portions at 9000 rpm to give 1.7 liters of supernatant with pH 6.8.

The supernatant is applied to a Dowex-lx2(C1-) 50100 mesh column, bed dimensions 3.9 cm.x25 cm., at a flow rate of 5-10 ml. per minute. The column is washed with 50 ml. of deionized water, followed by 2 liters of 0.15 M Nail+0.02 M Tris-HC1 buffer, pH 7.0, +25 ,uM neutral EDTA in 50% (v/v) aqueous methanol.

The product is then eluted with 30 g/.liter Nail+0.02 M Tris-HC1 buffer, pH 7.0+25 MM EDTA in 80 ,U, (v/v) aqueous methanol, at a flow rate of 5 ml./minute.

Fractions of 10 ml. are collected.

Bioactivity appears in fractions 5 to 180, with a maximum at fractions 25 to 70.

Fraction 12 to 105 are combined and concentrated to 15 ml. under reduced pressure. The pH is adjusted to 6.5 with 1 M HCI, and the concentrate is applied to a column (3.4x 55 cm.) of XAD-2 which has been washed with 2.5 liters each of 60 (v/v) aqueous acetone, deionized water, and 5% NaCI in deionized water. The column is rinsed with 3x5 ml. portions of deionized water and is eluted with deionized water at 10 ml./minute. Fractions of 10 ml. are collected.

Bioactivity appears in fractions 32 through 270 with a maximum at fractions 42 to 62. the total bioactivity eluting is 25% of the activity of the original broth.

Fractions 40 to 240 are combined, and the stored combined fractions 12 to 34 from the XAD-2 column of Example 1 are added. The total combined fractions are concentrated under reduced pressure to 50 ml., containing 120 HAEA304 units.

The concentrate is diluted with 200 ml. methanol, and applied at 2 ml./minute on a column (2.15x40 cm.) of Dowex-lx4, minus 400 mesh, which has been previously washed with 2 liters of 30 g./l. NaCI in 80% aqueous methanol, and 1 liter of 80% aqueous methanol. After application of the sample, the column is washed with 100 ml. of 80% (v/v) aqueous methanol and is eluted with 2.5 liters of 0.22 M Nail+0.01 M NH4Cl+0.0002 M NH3 in 80% (v/v) aqueous methanol, followed by 3 liters of 0.31 M Nail+0.01 M NH4Cl+0.0002 M NH3 in 80% (v/v) aqueous methanol, The flow rate is 2 ml./minute, and fractions of 10 ml. each are collected.

Antibiotic 890at is separated on the Dowex-lx4 column eluted in fractions 280 to 350, as measured by assay on ATCC 8461.

Dowex-lx4 fractions 307 to 338 combined and concentrated under reduced pressure to 7.6 ml., and 30,us. of I M NaOH are added to bring pH into the range 7.0 to 7.5. The concentrate is diluted with 7.6 ml. of methanol, and the solution is centrifuged to remove the salt precipitate. The supernatant is concentrated under reduced pressure to 4 ml., and to this concentrate, 6 ml. of methanol are added.

The salt precipitate is allowed to settle and this supernatant is pipetted onto a column (2.2x70 cm.) of Sephadex-G10, which has been previously washed with 10 ml. of 1 M NH3 in 80% aqueous methanol followed by I liter of 0.0001 M NH3 in 80% methanol. After application of the sample, the column is rinsed three times with 1 ml. of 0.0001 M NH3 in 80 methanol, and is washed with 400 ml. of the same solvent at a flow rate of 1 ml./minute. The column is then eluted with 0.00005 M NH3 in 50% aqueous methanol at a flow rate of I ml. per minute, and 10-ml.

fractions are collected, with numbering starting from the first application of the 5Q7 methanol eluate.

A peak of absorbance at 305 nm appears in fractions 9 to 22 with a maximum at fraction 12. Fractions 11 to 17 are combined, containing 17 A305 units, of which 9.1 are hydroxylamine-extinguishable.

The combined fractions are concentrated to 2 ml. under reduced pressure; are diluted to 10 ml. with 99.7% D2O, and are then concentrated to 1.5 ml. under reduced pressure, and frozen and lyophilized. The resulting white powder, consisting of product 890at and more than 5 mg. of sodium chloride, is analyzed by NMR spectroscopy.

EXAMPLE 4 Antibiotic 890A1 A tube of lyophilized culture of Streptomyces flavognseus MA-4600 NRRL 8140 is aseptically opened and the contents suspended in a tube containing 1.5 ml.

of sterile Medium E having the following composition.

Medium E Yeast Extract 10.0 g.

Glucose 10.0 g.

MgSO4. 7H2O 0.05 g.

Phosphate Buffer** 2 ml.

Distilled H2O 1000 ml.

**Phosphate buffer solution: KH2PO4 91.0 g.

Na2HPO4 95.0 g.

Distilled H2O 1000 ml.

This suspension is used to inoculate a 250-ml. triple-baffled Erlenmeyer seed flask containing 54 ml. of seed Medium C having the following composition.

Medium C Autolyzed Yeast (Ardamine*) 10.0 g.

Glucose 10.0 g.

MgSO4 7H2O 0.05 g.

Phosphate Buffer** 2 ml.

Distilled H2O 1000 ml.

pH adjusted to 6.5 with NaOH *Ardamine: Yeast Products Corporation **Phosphate buffer solution: KH2PO4 91.0 g.

Na2HPO4 95.0 g.

Distilled H2O 1000 ml.

The seed flask is stoppered with cotton and shaken for 30 hours at 280C+ 10C. on a 220 rpm gyrotory shaker, 2-inch throw.

Fifty 250 ml. unbaffled Erlenmeyer production flasks, each containing 40 ml. of production Medium F are inoculated with I ml. per flask of the broth from the seed flask. The production flasks are stoppered with cotton.

Medium F Tomato Paste 20.0 g.

Primary Yeast 10.0 g.

Dextrin (Amidex) 20.0 g.

CoCI2 6H2O 5.0 mg.

Distilled H2O 1000 ml.

pH adjusted to 7.2-7.4 with NaOH After inoculation, the production flasks are incubated at 280C.110C. with shaking on a 220 rpm gyrotory shaker, 2-inch throw for three days. The flasks are assayed for activity against standard Vibrio percolans ATCC 8461 assay plates using 1/2 inch assay discs dipped into centrifuged fermentation broth samples. Samples are diluted with 0.05 M phosphate buffer, pH 7.4. The results are tabulated below.

Harvest Age Hours 72 pH 6.4 Vibrio percolans (1/100 Dilution) Assay 23 mm.

890 Assay, units/ml. 103 The whole broth is centrifuged in 200 ml. portions in polycarbonate bottles at 9000 rpm for 15 minutes to give 1600 ml. of combined supernatants with a potency of 104 units/ml. To this is added 0.5 ml. of 0.1 M neutral EDTA.

The centrifuged broth is adsorbed on a Dowex-lx2 (Cl-), 5010ü mesh column, bed dimensions 3.8x22 cm., at a flow rate of 6 to 20 mlymin. The column is rinsed with 100 ml. of deionized water and eluted with I liter of deionized water dimensions 2.2x41 cm., at a flow rate of 2 ml./min. the column is rinsed with 50 ml.

of deionized water, and eluted with 3 liters of 0.07 M Nail+0.005 M NH4Cl+0.0001 M NH3 in deionized water, at a rate of 2 ml./min. Fractions of 10.8 ml. are collected, starting from the first application of eluent.

The main peak of antibiotic 890A, appears in fractions 181 to 217, with a maximum at fraction 198. Fractions 186 to 210, containing a total of 114 absorption units at 300 nm., are pooled The pooled fractions are concentrated to 4.0 ml., and the pH is adjusted to 7.3 by addition of 16 u liter of 1 M NaOH. The concentrate is applied on a column of Bio-Gel P-2, 200400 mesh, bed dimension 2. 15x70 cm., and is washed in with 3x 1 ml. washes of deionized water and eluted with deionized water at 0.96 ml./min.

Fractions of 3.85 ml. are collected.

The main peak of antibiotic 890A, appears in fractions 24 through 44, with a maximum at fractions 33 and 34. Fractions 27 through 38, having the highest A30JA24s ratios, are combined for lyophilization. These combined fractions have a total of 72 A300 units.

To carry out the lyophilization, the combined fractions are concentrated to 3.0 ml. and the pH of the concentrate is adjusted to 7.5 by addition of 10 y liters of 0.1 M NaOH. The sample is divided into two portions of 1.50 ml. each, and the portions are separately quick-frozen and lyophilized from 14 ml. glass screw-cap vials. Each sample contains 1.73 mg. of 890A1, corresponding to 35.8 A300 units.

WHAT WE CLAIM IS: 1. A method of producing the compound Antibiotic MSD 890at, which has the following formula:

that comprises cultivating a 890Ag-producing strain of Streptomvees flavogriseus in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions and recovering said antibiotic.

2. A method as claimed in Claim 1, in which the micro-organism cultivated in Streptomyces tlavogriseus NRRL 11,020.

3. A method as claimed in Claim 1, carried out substantially as hereinbefore described in any one of the Examples.

4. Antibiotic MSD 890A9 when prepared by a method as claimed in any one of

Claims (1)

1. Claims 1 to 3.

   5. A pharmaceutically acceptable salt of a compound as claimed in Claim 4.

   6. A composition comprising an antibacterial effective amount of a compound as claimed in Claim 4 or 5 and a non-toxic pharmaceutically acceptable carrier.

   7. A composition as claimed in Claim 6 in the form of an animal feedstuff.

   8. A composition as claimed in Claim 6 in the form of an aqueous composition containing 0.1 to 100 ppm of the said compound.

   9. A composition as claimed in Claim 6 in the form of an intramammary preparation.

   10. A composition as claimed in Claim 6 in the form of an injectable solution or suspension.

   11. A composition as claimed in Claim 6 in the form of a tablet or capsule.