CH634104A5 - Process for the preparation of the novel antibiotic MSD 890A10 - Google Patents

Description

634104

2nd

PATENT CLAIMS 1. Process for the preparation of the new antibiotic MSD 890Aio in the form of the compound of the formula

OSO-H

I 3

0

CH3 ~ CH '

II

S-CH2-CH2-NHC-CH3

0

X

COOH

or their pharmaceutically acceptable salts, thereby growing and winning the antibiotic.

characterized in that one is an 890Aio-forming strain of 2. The method according to claim 1, characterized in that Streptomyces flavogriseus in an aqueous nutrient medium that the cultured microorganism is Streptomyces flavogri-the assimilable source of carbon, nitrogen and inorganic NRRL11 020.

contains ganic salts, under submerged, aerobic conditions

The present invention relates to a method turai Research Service, U.S. Department of Agriculture, Peo-for the manufacture of the new antibiotic MSD 890Aio (hereafter 25 ria, III, deposited and is referred to under the accession number as “Antibiotic 890Aio”), which is both available under NRRL 11 020.

against gram-positive as well as against gram-negative Streptomyces flavogriseus MA4638 shows that antibiotic bacteria are active. The antibiotic is obtained by culturing kum 890Aio, which is isolated essentially in pure form from the Fer species of Streptomyces on suitable fermentation broth.

serve. 30 The morphological and cultural characteristics of

The finding of the remarkable antibiotic Streptomyces flavogriseus MA-4638 are in the following

Properties of penicillin has shown great interest on the table.

stimulates this area. As a result, many other valuable, antibiotic substances were found. In general, the morphology includes the antibacterial activity of each of these antibiotics 35 The spore carriers are branched, straight-chain to undetermined, clinically important pathogenic bacteria. In spur chains, which form tufts. The chains are, for example, some are only more than 10 spores long compared to gram-posi. The spores are spherical to oval -

active types of bacteria. The resulting resistance 0.9x 1.2 \ x. (970x).

in the course of widespread use of the existing ones

Antibiotics in the treatment of bacterial infections have caused 40 cultural properties to create a serious resistance problem. Oatmeal agar (ISP Medium 3): vegetative growth -

Accordingly, the disadvantages of the known anti-reverse yellow-dark yellow lined with brown, ruffled; biotikadie further research for other antibiotics stimu- Luftmycelium - light gray lined with medium gray; soluble pigments that are sensitive to a wide range of pathogens - none;

as well as against resistant strains of special microor- 45 Czapek Dox Agar (sucrose nitrate agar): vegetative ganisms are active. Growth - reverse-brown lined with dark brown; Air-

The invention comprises a method for producing the mycelium - medium gray, velvety; soluble pigment - light antibiotic in diluted form as a raw concentrate and in browning of the medium;

pure form. Egg albumin agar: vegetative growth - reversed-yellow-

The invention is therefore based on the aim of a method 50 lined with dark yellow with brown; Aerial mycelium - medium gray, used to produce a new and valuable antibiotic mixed with yellowish gray (2dc) and greyish yellow (2db); to show the pigment which is soluble in the inhibition of growth - light yellowish dark yellow; Glycerin Aspa-ner gram-negative and gram-positive microorganisms raginagar: vegetative growth - reverse brown; Luftmy-

is highly effective. It is supposed to be a process for producing the celium - velvety, light gray with yellowish tone (2dc); soluble new antibiotic substance by fermentation of nutrient 55 pigment - light dark yellow;

media created with Streptomyces species. Inorganic salt starch agar (ISP Medium 4) vegetarian

The new antibiotic compound is grown by growing ves - reverse-greenish-yellowish-dark yellow; Luft - a new strain of Streptomyces flavogriseus near mycelium - velvety, medium gray with a yellow tone (3fe); solvable-controlled conditions. pigment - very light dark yellow;

Based on extensive taxonomic studies of yeast extract dextrose + salt agar: vegetative growth became the strain of the microorganism, which in the present case - - reversed-dark brown; Aerial mycelium - dark gray, the invention was used as to the streptomy species - mixed with a lighter gray; soluble pigment - none; ces flavogriseus properly identified and in the Hinterle yeast extract malt extract agar (ISP Medium 2): vegetative gene from Merck & Co., Inc., Rahway, N.J., as MA4638 growth - reverse brown; Luftmycelium - velvety, darkly labeled. Its culture was lined perma gray on September 15, 1976 with a lighter gray; soluble pigment -nent with no restriction on availability at the none;

Northern Regional Laboratories depository, Nor Skim Milk Agar: vegetative growth - dark yellow; thern Utilization Research and Development Division, Agricul- Luftmycelium - sparse, whitish; soluble pigment - light

Browning of the medium; Hydrolysis of casein - good;

Litmus milk: vegetative growth - moderate ring growth, dark yellow; Aerial mycelium - none; Color - purple; Coagulation and / or peptonization - complete peptonization; Becoming alkaline;

Skim milk: vegetative growth - moderate ring growth, dark yellow; Aerial mycelium - none; soluble pigment - light brown; Coagulation and / or peptonization - complete peptonization; Becoming alkaline;

Nutrient tyrosine agar: vegetative growth - reversed dark brown; Aerial mycelium - dark gray lined with grayish white; soluble pigment - slight browning of the medium; Decomposition of tyrosine - positive;

Peptone-iron-yeast extract agar: vegetative growth -dark yellow; Aerial mycelium - whitish, moderate; soluble pigment - none; Melanin - none; HîS formation - negative;

Nutrient agar: vegetative growth - reverse-light grayish brown; Aerial mycelium - light gray lined with dark gray; soluble pigment - none;

Nutrient strength agar: vegetative growth - dark yellow lined with gray; Aerial mycelium - medium gray; soluble pigment - none; Starch hydrolysis - good;

Nutritional gelatin agar: vegetative growth - dark yellow lined with gray; Aerial mycelium - grayish-white; soluble pigment - none; Liquefaction of gelatin - good;

Potato sting or cone: vegetative growth - dark yellow; 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 tint: vegetative growth - dark yellow; Aerial mycelium - none; soluble pigment - none; Liquefaction of gelatin - completely.

All readings above are made after three weeks of incubation at 28 ° C, unless otherwise stated. The pH of the media used in these tests is approximately neutral, namely pH 6.8 to 7.2. The color names used in the description correspond to the definitions of Color Harmony Manual, 4th Ed. (1958), Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus MA-4638 is also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism is grown on a synthetic base medium (Pridham and Gottlieb) containing 10% carbohydrates for 3 weeks at 28 ° C. The pH of the media used in these tests is approximately neutral (6.8 to 7.2). Table I lists the utilization of these carbohydrate sources by Streptomyces flavogriseus MA4638, where + growth, ± bad or questionable growth and - mean no growth compared to the negative control (no carbon source).

Table I

glucose

+

Maltose

+

Arabinose

+

Mannitol

+

Cellulose

-

Mannose

+

Fructose

+

Raffinose

-

Inositol

-

Rhamnose

+

Lactose

+

Sucrose

±

Xylose

+

The strength of growth will change with the temperature and oxygen demand of the microorganism as follows:

634104

Temperature range (yeast extract - dextrose + salt agar) 28 ° C - good vegetative growth and air growth 37 ° C - good vegetative growth; no air hyphae 50 ° C - no growth.

Oxygen demand (stub culture in yeast extract dextrose + salt agar) aerobic.

The present invention is not limited to the organism Streptomyces flavogriseus or to organisms that fully meet the above growth and microscopic properties; these are listed for illustration only. The present invention is also intended in particular to include the use of mutants which are formed from the organism described by various means, such as X-ray radiation, UV radiation, nitrogen mustard, exposure to bacteria, and the like.

890Aio is formed during aerobic fermentation under controlled conditions of suitable aqueous nutrient media that are inoculated with a strain of the organism Streptomyces flavogriseus. Aqueous media, such as those used to form other antibiotics, are suitable for the formation of 890Aio. Such media contain sources of carbon, nitrogen and inorganic salts which are assimilable by the microorganism.

In general, carbohydrates such as sugar, e.g. Dextrose, glucose, fructose, maltose, sucrose, xylose, mannitol and the like, and starches such as dextrin or granules or grains such as e.g. Oats, rye, corn starch, corn flour and the like, either alone or together with sources of assimilable carbon, can be used in the nutrient medium. The amount of carbohydrate normally varies between about 1 and 6% by weight based on the medium. These carbon sources can be used individually or several such carbon sources can be mixed or combined in the medium. In general, many proteinaceous materials can be used as nitrogen sources in the fermentation process. Suitable nitrogen sources include e.g. Yeast hydrolysates, primary yeast, soybean meal, cottonseed meal, hydrolysates of casein, corn steep liquor, soluble substances from the production of brandy or stillage, etc.; the preferred source are soluble substances that are obtained during the production of brandy or stillage. The sources of nitrogen, either alone or together, are used in amounts ranging from about 0.2 to 6% by weight based on the aqueous medium.

Among the inorganic nutrient salts that can be incorporated into the culture media are the usual salts that give sodium, potassium, ammonium, calcium, magnesium, phosphate, sulfate, chloride, carbonate and similar ions. Trace metals such as cobalt, manganese and iron are also included.

The media described in the examples are only examples of a wide variety of media that can be used.

The fermentation is expediently carried out at a temperature in the range from 20 to 37 ° C .; however, for optimal results it is preferred to carry out the fermentation at a temperature of 23 to 28 ° C. The initial pH of the culture media suitable for the growth of the strains of Streptomyces flavogriseus culture and for the formation of the antibiotic 890Aio can vary from 6.0 to 8.0.

Small-scale fermentation of the antibiotic is conveniently carried out by inoculating a suitable nutrient medium with the culture providing the antibiotic and, after transfer into the production medium, allowing the fermentation to proceed for several days at a constant temperature of about 24 ° C on a shaker.

The fermentation is carried out in a sterilized flask of nutrient medium through one or more stages of the inoculation material3

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634104

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development initiated. The nutrient medium used for the vaccination stage can be any suitable combination of carbon and nitrogen sources. The inoculation flask is shaken in a constant temperature chamber at about 28 ° C for one day or until sufficient growth and part of the resulting growth is used to inoculate either a second stage inoculum or the production medium. Flasks with intermediate vaccine material, when used, are similarly developed, i.e. part of the flask content from the last inoculation stage is used to inoculate the production medium. The inoculated flasks are shaken at constant temperature for several days and the contents of the flask are centrifuged or filtered towards the end of the incubation period.

When working on a large scale, it is preferred to carry out the fermentation in suitable tanks which are equipped with a stirring device and a device for venting the fermentation medium. According to this method, the nutrient medium is prepared in a tank and sterilized by heating at a temperature up to about 120 ° C. After cooling, the sterilized medium is inoculated with the previously grown inoculum of the producing culture and the fermentation can be carried out for a period of e.g. 1 to 6 days with stirring and / or aeration of the nutrient medium and maintaining the temperature at about 22 to 26 ° C. This method of producing the 890Aio antibiotic is particularly suitable for the manufacture of large quantities of antibiotics.

Physical and chemical properties of the antibiotic 890Aio

Antibiotic 890Aio is an acidic compound that migrates towards the positive pole during electrophoresis at neutral pH. With a gradient of 50 V / cm in 0.03 M potassium phosphate buffer, pH 7.1, the antibiotic moves 8.0 cm in 30 min compared to a 4.0 cm movement for 890Ai. The disodium salt is a white or light yellow powder after lyophilization from an aqueous solution. Under acidic conditions in aqueous solution, the antibiotic is unstable and the free acid form has not been isolated.

The disodium salt of the antibiotic 890Aio has an absorption maximum at 299 nm and a minimum at 243 nm at neutral pH in water. The E% at 300 nm of the best purified preparation of the disodium salt is 214. The A300 / A250 ratio is 3.33 for the purest sample and the A300 / a220 ratio is 2.05. The presence of minor impurities in this sample suggests

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35

that the corresponding ratios would be somewhat higher for a sample with complete purity. More than 94% of the absorption at 300 nm can be extinguished by reaction with hydroxy-lamin at neutral pH. The absorption at 250 nm also decreases when reacting with hydroxylamine, and the ratio of the absorption decrease at 250 nm to the decrease at 300 nm is approximately 0.16. The reaction with hydroxylamine, which is followed by the A3oo decrease under the conditions described in the section “Hydroxylamine Reaction”, appears to be first order, with a half-life at room temperature of 23 to 60 seconds.

Determined against a standard of the antibiotic 890Ai, the antibiotic 890Aio has 174 bioassay units per HAEA3oo unit. HAEA300 is described under the heading “Hydroxylamine Reaction”.

Table II shows the 100 MHz signals in the nuclear magnetic resonance spectrum of 890Aio in d2o at 32 ° C. The chemical shifts are given in ppm, relative to HOD, at 4.70 S at 32 ° C and the coupling constants are given in Hertz.

Tables cfchch chsco c (6) -h c (5) -h c (8) -h c (,) - h2

-ch2nh -ch2-S-

1.55 (3H, D, 6.5 Hz)

2.02 (3H, S)

3.89 (1H, D, D, J6.5 = 5.4 Hz, J6.8 = 9.2 Hz)

- 4.34 (1H, D, T, J5-6 = 5.5 Hz, j5-1 = 9.5 Hz)

- 4.80 (partially covered by HOD line)

- 3.14 (1H, D, D, ~ 9.2 + ~ 18 Hz)

- 3.33 (1H, D, D, ~ 10 + 18 Hz)

3.43 (2H, T, 7 Hz)

3.03 (2H, M)

The mass spectrum of TMSi-890Aio is characterized by the fragments listed in Table III.

Table III

m / e 86

227.0224

241

300/1

339.1325

342.1444

368

440

458

C5H15SO4SÌ2, calculated 227.0230

C13H25NO4SÌ2, calculated 339.1322 C15H26N2O3S Si, calculated 342.1433

door:

The 890Aio antibiotic has the following molecular structure

oso ^ h ch3-Ìh ooh s-ch2-ch2

(I)

?

-nh-c-ch-

The antibiotic 890Aio is further characterized by the following biotics on the surface of a 100x 15 mm petri dish, which distinguishes the antibiotic spectrum profiles. 65 contains 5 ml of inoculated nutrient agar plus 0.2% yeast extract,

The test for the determination of the antibiotic spectrum is determined by incubating at 25 ° C. The results, from the file of the antibiotic 890Aio, are expressed in Table IV by the application of a depressed diameter of the inhibitor or Inhi 0.015 ml of a 9.5 ng / ml aqueous solution of the anti-zone.

5

634104

Table IV

Organism zone of inhibition,

Diameter, mm

Bacillus sp. MB No. 633 30

Proteus vulgaris MB No. 1012 19

Pseudomonas aeruginosa MB No. 979. 0

Serratia marcescens ATCC 890 15

Staphylococcus aureus ATCC 6538P 20

Bacillus subtilis ATCC 6633 30

Sarcina lutea ATCC 9341 33

Staphylococcus aureus MB No. 698 23

Streptococcus faecalis MB No. 753 0

Alcaligenes faecalis ATCC 213 24

Brucella bronchiseptica ATCC 4617 0

Salmonella gallinarum MB No. 1287 28

Vibrio percolans ATCC 8461 33

Xanthomonas vesicatoria MB No. 815 20

Proteus vulgaris ATCC 21100 30

Escherichia coli MB No. 1418 25

Pseudomonas stutzeri ATCC 11607 0

Klebsiella pneumoniae MB No. 1264 19

Aerobacter aerogenes MB No. 835 22

Erwinia atroseptica ATCC 4446 10

Pseudomonas aeruginosa MB No. 2824 0

Corynebacterium pseudodiph. ATCC 9742 12

Escherichia coli ATCC 9637 17

Streptococcus faecium MB No. 2820 0

Strep'ococcus agalactiae MB No. 2875 29

Vibrio percolans MB No. 2566 (res. Ceph C) 22

Proteus vulgaris MB No. 2112 (Episom) 30

Proteus mirabilis MB No. 3126 29

Vibrio percolans ATCC 8461 + 2 x 10S jx / ml 25 penicillinase

Vibrio percolans ATCC 8461 + β-lactamase from 34 Enterobacter clacae MB 2646

Antibiotic 890Aio is active against various gram-positive and gram-negative bacteria and it is a potent inhibitor of bacterial β-lactamases; it can be used in human and veterinary medicine. 890Aio can be used alone or together with other antibacterial drugs for the treatment of infections caused by gram-positive or gram-negative bacteria, e.g. against Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae and Salmonella schottmuelleri.

The described compound can be used together with β-lac-tam antibiotics which are sensitive to β-lactamases to inhibit the action of such β-lactam antibiotics by inhibiting the lactamase activity and thus the useful life of the Antibiotics are extended.

A mixture of antibiotic 890Aio with a lactamase-sensitive β-lactam antibiotic will be more effective in treating infections with β-lactamase-producing bacteria than the same amount of β-lactamase-sensitive antibiotic alone.

The antibiotic 890Aio can be used as an additive for animal feed, for the preservation of food and feed and as a disinfectant. It can be used in aqueous compositions in the range of about 0.1 to about 100 parts of antibiotic / million parts of solution or preferably in concentrations in the range of about 1 to about 10 parts of antibiotic / million parts of solution to destroy and inhibit the growth of harmful bacteria on medical and dental devices or devices and used as bactericides in industrial applications.

The 890Aio antibiotic can be used in pharmaceutical preparations as the only active ingredient or together with one or more other antibiotics or with one or more pharmacologically active substances.

The antibiotic can be administered orally, topically, intravenously or intramuscularly. The methods used for administration can be any of the methods conventional in the art or any method that can be adapted to the antibiotic described herein.

In addition to a carrier, the preparations described can contain other ingredients, such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, viscosity agents or flavoring agents, as are well known and normally used.

In veterinary medicine, such as in the treatment of 20 poultry, cattle or cows, sheep, pigs and the like, the preparations can e.g. in the form of intramammary preparations, either in long-acting or rapidly releasing base materials.

The dose to be administered depends on a large scale 25 on the condition of the subject to be treated, the weight of the host and the type of infection, the route and the frequency of the administration. The parenteral route is preferred for generalized infections and the oral route is preferred for intestinal infections.

In the treatment of bacterial infections in humans or living beings, the described compounds can be administered simultaneously with a β-lactamase-sensitive antibiotic orally or parenterally according to methods known per se for the administration of antibiotics. The anti-35 biotic 890Aio is administered in an amount of about 2 to 600 mg / kg / day and preferably in an amount of about 5 to 100 mg / kg / day, preferably in a divided dose, e.g. three or four times a day. It can be administered in dosage units, e.g. Contain 100,330,400 or 1000 mg of active ingredient 40 with suitable, physiologically acceptable carriers or diluents. The dose units are in the form of liquid preparations, such as solutions or suspensions, or as solids in tablets or capsules. The optimal dose administered in any particular case will, of course, depend on the type and severity of the infection to be treated. Smaller doses are used in pediatrics, with all of these settings and adjustments being familiar to the person skilled in the art.

The method according to the invention also includes the preparation of the non-toxic, pharmaceutically acceptable salts of 890Aio, e.g. the pharmacologically acceptable salts formed with inorganic and organic bases, for example metal salts derived from alkali metal or alkaline earth metal hydroxides, carbonates or bicarboxylic acids, such as those derived from sodium, potassium, ammonium and calcium, and salts which are derived from primary, secondary or tertiary amines, such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower alkylene diamines, N, N-diaral-60 alkyl lower diamines, aralkylamines, amino-subst . -Low-rig-alkanols, N, N-di-lower-alkylamino-subst.-lower-alkano-len, amino-polyamino and guanidino-subst-lower-alkanoic acids and nitrogen-containing heterocyclic amines. Specific examples are 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,

634104

6

Morphine, benzylamine, ethylenediamine, N, N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-l-propanol, theophylline, N-methylglucamine and the like. deduce.

The salts of the described compound can either be isolated directly from the fermentation medium using suitable eluents during ion exchange chromatography, or they can be prepared by processes known per se. For example, the disalts, such as the disodium salt, can be treated by treating 2 equiv. Sodium hydroxide can be prepared with 1 mole of product (I) in a suitable solvent. Mixed salts with monovalent cations can be prepared by mixing 1 mol of a monovalent base with 1 mol of product (I) plus 1 equiv.

another base. Alternatively, monobasic salts can be treated by treating 1 equiv. a base with a monovalent cation can be prepared with 1 mol of product (I). The salts can also be prepared by treating 1 mole of the product with 1 mole of a base with a divalent cation. The salts according to the invention are pharmacologically acceptable, non-toxic derivatives which can be used as active constituents in suitable pharmaceutical forms in dosage units. They can also be made with other drugs to produce preparations with a wide range of activities.

Fermentation broths which contain the antibiotic 890Aio and which have been produced by the processes according to the invention have activities in the range from about 2 to 170 units / ml when they are processed according to the disk diffusion assay or. analysis methods using Vibrio percolans (ATCC 8461). The antibiotic 890Aio contained in these fermentation broths can be isolated and purified by a number of methods. In such a process, the 890Aio antibiotic is adsorbed on a strongly basic anion exchange resin. Examples of such strongly basic anion exchange resins are those with a styrene-divinylbenzene matrix, e.g. the polystyrene resin "Dowex" 1x2 with quaternary ammonium groups on the core (manufactured by Dow Chemical Co., Midland, Michigan) in the chloride cycle. Other examples of this class of strongly basic exchange resins include the following: "Duolite" A-40, A-42, A-101, A-102 and A-14 (manufactured by Chemical Process Co., Redwood City, California); Amberlite IRA-400, IRA-401 and IRA-410. Alternatively, a weakly basic anion exchange resin such as "Amberlite" IRA-68 can be used. (Amberlite resins are manufactured by Rohm and Haas, Washington Square, Philadelphis 5, Pennsylvania.)

The adsorbed antibiotic can be easily eluted from the anion exchange resin with salt solutions in 80% (vol / vol) aqueous methanol. The eluate obtained in this way can be further purified by other purification methods. The eluate can be purified by concentrating and passing it through a column packed with polystyrene, a non-polar, hydrophobic, cross-linked divinylbenzene polymer such as XAD-1,2 and 4, or with polyacrylamide resins such as XAD-7 and 8. XAD-2 is preferred. (XAD-1,2,4,7 and 8 are manufactured by Rohm and Haas, Philadelphia 5, Pennsylvania.)

One method of producing further purified 890Aio antibiotic is the use of gel filtration through polyacrylamide gel with a pore size that excludes molecules with a molecular weight above 1800, such as "Bio-Gel" P-2 (manufactured by Bio. Rad, Richmond , California). Other gels, such as "Sephadex" G-10, can also be used for desalination.

The preferred method according to which the antibiotic 5 890Aio is obtained in high purity from a broth is to centrifuge or filter the broth to remove the solids; the adsorption and elution of the filtrate from an anion exchange resin, such as "Dowex" -l x2, in a chloride cycle with 3% NaCl in 80% (vol / vol) aqueous methanol, io whereby the antibiotic is both concentrated and partially purified at the same time; Pass through a column of appropriately prepared XAD-2, which retains the antibiotic and the «Dowex» -1 x2 eluate is cleaned and desalted. The fractions i5 enriched with 890Aio are combined and further purified. Chromatography on a «Dowex» -l x2, particle size max. 0.037 mm, resin eluting with NaCl and / or NH4CI in 80% aqueous methanol gives a product that has been freed from many of the original UV absorbing contaminants 20 (the NH4CI is used to achieve some buffering capacity in the eluent ). The 890Aio is separated from other antibiotics. The majority of the salt introduced in the «Dowex-l» x2-Chromato-25 graphie is removed by desalting with «Bio-Gel» P-2 or «Sephadex» G-10.

The remaining impurities can be removed by additional chromatography cycles on “Dowex-l” x2, (-0.037 mm) with elution through a solution containing sodium chloride and 30% methanol, followed by desalting. 30 In the purification by column chromatography, generally only those fractions of the eluted volume which contain the antibiotic at least 30% as pure as the purest fraction are combined for further purification. The criteria for the purity are the ratios bioactivity / 35 a220, A300 / A250 and HAEA300 / 22O and the conductivity in the desalination process. For each chromatography level, a220, A2S0, A300 and the bioactivity of suitable fractions are therefore determined. If possible, HAEA300 is also measured and the conductivities are determined during desalination. The criteria for deciding which fractions are combined for the subsequent treatments can be set somewhat so that one obtains higher yields at the expense of purity and vice versa a higher purity at the expense of yield.

45 When working on a laboratory scale (less than 201 sample volumes), all chromatography steps, with the exception of XAD-2 chromatography, are carried out in a cold room at 2 to 5 ° C. XAD-2 chromatography is carried out at room temperature. The pH of the anti-50 biotic solutions to be stored is adjusted by carefully adding dilute NaOH or HCl solutions. Aqueous solutions are stored in a refrigerator or preferably in ice water and 50 to 80% methanol solutions at -20 to -60 ° C.

55 In the purification steps prior to XAD-2 chromatography, the solutions are generally adjusted to 25 p, M in EDTA by adding Vaooo volume of a solution of 0.1 M Na2 EDTA, which is brought to a pH of 7.0 Addition of sodium hydroxide (0.1 M "neutral EDTA") was neutralized 60.

Below is a flow chart for the cleaning process used to make the 890Aio antibiotic.

7

890Aio antibiotic cleaning procedure

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Fermented broth containing 890A-J0

1. Cool

2nd center

Centrifuged broth

1.Adsorb. on "Dowex" -lx2 (Cl) (0.297-0.149 mm)

2. Wash with 0.15M NaCl + 0.01 M

fugieren 3.Zugabe v

EDTA

tris-HCl + 25 / UM EDTA in 50 # MeOH '

3. Wash with water

4. Elute with 3% NaCl + 0.01 M tris-HCl, pH 7, + 25 / UM neutral.

v EDTA in 8056 methafcol

"Dowex" -lx2>

80 »0H

Eluate

Ì 1. Concentrate

2. Adsorb to XAD-2

3.Elution with water

XAD-2 eluate

1. Concentrate the fractions containing 890A> jo

2.Dilute with MeOH _

3.Adsorb. on "Dowex" -lx4 (Cl) (0.037 mm)

^ .Eluting with 0.26M NaCl + V 0.005M NHaCl + 0.00005M NH3 in 80% methanol

"Dowex" -lx4 eluate 890A10

1.Dilute with water _

2.Adsorb. on "Dowex" -lx2 (Cl) (0.037 mm)

3.Eluting with 0.26M NaCl + 0.005M NH4C1 + 0.00005M NH,

w in 30% methanol D

"Doi * ex" -lx2 eluate

1. Concentrate

2. Adsorb to "Bio-Gel" P-2

3. Elute with 0.02 mM NH ^

"Bio-Gel" P-2 eluate

1. Concentrate

2. Lyophilize

634104 8

Analysis or assay procedure for antibiotic 890Aio mm adopted and (potency of the standard) is considered as 1

I. Bioassay or analysis unit / ml assumed, determined with Vibrio percolans ATCC

An agar plate disk diffusion method is used under 8461. Pure 890Ai is used as such with a potency of 250 using Vibrio percolans ATCC 8461 as test organism units per absorbent unit which can be extinguished by hydroxylamine. A purified sample of the antibiotic 890Ai 5 th defined at 300 nm when used as the standard is used as the standard. The 890Ai antibiotic is prepared according to the procedure described below. Analysis procedures for determining the 890 assay units. ten »

Plates containing Vibrio percolans ATCC 8461 are manufactured according to an agar plate disk diffusion method known per se. io drive using Vibrio percolans ATCC

A lyophilized culture of Vibrio percolans ATCC 8461 8461 was used as the test organism. Cephaloridine is suspended in 15 ml of a sterilized medium using 8 g / l standard. The Vibrio percolans ATCC 8461 containing Difco nutrient broth and 2 g / 1 yeast extract in distilled water plates are prepared as follows. A culture of contains "nutrient broth yeast extract" (hereinafter referred to as NBYE Vibrio percolans ATCC 8461 in nutrient broth yeast extract). The culture is incubated overnight on a rotary is overnight on a rotary shaker at 28 ° C shaker at 28 ° C. This culture is incubated and then used to a density of 60% permeable inoculation of the surface of oblique cultures, diluted at 660 nm. A 33.2 ml portion of this diluted the 1.5% agar contained in NBYE. The inoculated slant culture is added to 11 of a medium which is incubated with nutrient cultures overnight at 28 ° C and then consists of a plus 0.2% yeast extract and is kept at 46 ° C. The refrigerator stored. 20 inoculated medium containing agar is in 100 x 15 mm

The chilled slant cultures, cast, chilled and philized culture from a single, 10 ml / dish lyophil Petri dishes, are kept up to 4 weeks at 2 to 4 ° C up to 5 days prior to use used in their manufacture as follows. An eyelet The concentration of cephaloridine that is equivalent to 1

of inoculum from the slant culture is dispersed in 50 ml NBYE, which is / ml 890Ai, by analysis on plates, as above in a 250 ml Erlenmeyer flask. Culture 25 described, but containing 5 ml of inoculated is overnight on a medium / plate rotary shaker, determined as follows. Four Konzentra-28 ° C incubated and then diluted to a density that a cephaloridine ions give the standard - 3,12,6,25,12,5

50% permeability at 660 nm results. A 33.2 ml portion of this and 25 (ig / ml, with 12.5 (ig / ml as a reference solution of genomic diluted culture) is added to 11 NBYE, which becomes 15 g. The zone diameter on a 5 ml plate for the

Contains agar and kept at 46 ° C. The inoculated, agar ent- 30 standard are as follows:

Holding medium is placed on 100 x 15 mm plastic petri dishes, 5 ml / dish, cooled and kept at 2 to 4 ° C. up to 5 concentration (ig / ml) zone diameter (mm) days before use. 3.12 16.8 filter paper disks with a diameter of 1.27 cm 6.25 22.3 are immersed in the solution to be analyzed and placed on the 35 12.5 25.0 agar. Alternatively, the disks can be loaded onto a dry disk by pipetting V10 ml of solution and then the disk can be placed on the agar. One unit is defined as the amount of antibiotic / ml

The diameter of the zone of inhibition is measured after a suitable incubation, which is a 25 mm zone of inhibition on a 5 ml plate, as in beer (12 to 24 h at 25 ° C.). If necessary, Part I can be described above. In this analysis, a dilution of the solutions to be examined in 0.05 M is therefore a concentration of 12.5 ng / ml of cephaloridine as equivalent to potassium phosphate buffer, pH 7.4, "potassium phosphate buffer" (in 1 unit of 890Ai / ml. Since the Slope of the line for the following referred to as KPB), or in deionized water cephaloridine is 4.0, the calculations are performed. Potency of a sample using a slope of 4.0.

The potencies or strengths are calculated as follows 45. An inclination is determined by the zone III. Hydroxylamine reaction diameter of a solution of antibiotic 890Aio and an antibiotic 890Aio reacts with hydroxylamine and gives four times the dilution (in KPB) of this solution. Two is a substance in which the absorption at 300 nm strongly slices of any concentration is on a single device. This provides a basis for the quantitative plate analysis and the average zone size at 50 of the 890Aio antibiotic.

every concentration is determined. The slope is equal to that. The solution to be analyzed is set to 0.05 M in potassium half of the difference in the average zone size. phat brought pH 7.4 by adding vio volume of a solution,

which contains 0.8 M K2HPO4. Then V100 volume of 1 M hydroxylamine hydrochloride are added and the absorption is determined at 300 nm in intervals of Vi to 2 min. The reaction is carried out at room temperature. First order kinetic values are assumed and the half-life from the decrease in absorption during the first 10 min is estimated. From this half-life, the point in time 60 is estimated after which no further decrease in absorption should be observed, and the observations are continued beyond this point in time. If no further decrease is observed beyond this point in time, the in which D is the average diameter of the total decrease in absorption (where, for the dilution zones known, Ds, the average diameter and the absorption of the hydroxylamine are corrected) The standard zones and “dilution” are the degrees or the absorption that can be extinguished as the “hydroxylamine at 300 nm thickness, to which the unknown is diluted prior to analysis (HAEA300)”. If the absorption was above this, mean. If no standard is used, if Ds decreases beyond 25 point in time, the rate of background

The potencies are calculated according to the following formula:

[D-Da] log 2

Potency (units / ml) = \ 1

(Potency of the standard) x dilution x 10

9

634104

sorption decrease calculated and the observed decrease at this time is corrected by the background decrease, assuming that the background decrease is linear over time. The corrected value is then recorded as HAEA300. 5

The number of HAEA3oo units is equal to the HAEA300, multiplied by the volume in ml.

The following examples explain the processes by which the products described are manufactured. These examples are provided for illustration only, and any modifications of the methods described, according to which the products are obtained, are to be considered analog methods and are encompassed by the present invention.

The manufacture of the 890Ai antibiotic is described in U.S. Application Serial No. 634,300. Expressis verbis is referred to this publication.

example 1

A slant culture containing MA-4638 is used to inoculate 250 ml Erlenmeyer flasks with baffles containing 50 ml medium.

Medium A dextrose

Yeast autolysate («Ardamine») * MgSOWHîO phosphate buffer **

distilled water pH 6, j

* "Ardamine": Yeast Products, Inc.

** Phosphate buffer solution:

KH2PO4

N32HP04

distilled water

10.0 g 10.0 g 0.05 g 2.0 ml 1000 ml

91.0 g 95.0 g 1000 ml

This flask is shaken at 28 ° C on a 220 rpm shaker, 5.08 cm vibration amplitude, for 2 days until growth is sufficient. After 2 days, this vaccine product is used to inoculate three 250 ml Erlenmeyer flasks containing 40 ml Medium B using 2 ml / flask (5%).

Medium B

Tomato paste or pulp of whole oats (ground)

distilled water pH: adjusted to 7.0 using NaOH

20.0 g 20.0 g 1000 ml table transferred to two inoculation flasks, each containing 500 ml of C medium. The inoculation flasks, 21, shaking flasks with three deflection elements, are equipped with a side arm which is closed with cotton or cotton wool.

C medium

Autolyzed yeast (Ardamine *) 10.0 g

Glucose 10.0 g MgS04.7H20 0.05 g phosphate buffer ** 2.0 ml distilled water 100 ml the pH is adjusted to 6.5 before sterilization with NaOH * "Ardamine": Yeast Products, Inc. ** Phosphate buffer solution :

KH2PO4 91.0 g

Na2HP04 95.0

distilled water 1000 ml

20th

30th

35

45

These production flasks are also shaken at 28 ° C. on a 220 rpm shaker for up to 4 days, with analytical tests being carried out during the fermentation. At 3 days of age, an aliquot of the supernatant solution from the centrifuged broth is used for classification and identification studies. Using 0.65 cm analysis disks on standard analysis plates, this broth gives a 22 mm zone of inhibition against Proteus vulgaris and a 15 mm zone of inhibition against Salmonella gallinarum. Bioautography of an electrophoretogram of the centrifuged broth shows two components. The faster moving, smaller spot contains 890Aio.

Example 2

Two frozen ampoules, each containing 2 ml of MA-4638 inoculum, are slowly thawed and the content becomes asep55

60

65

The inoculated inoculation flasks are shaken for 24 to 30 hours at 28 ° ± 1 ° C on a 210 rpm gyrotation shaker, 5.08 cm vibration amplitude. The growth from the inoculation flasks is used to inoculate two 141 glass fermentation containers containing 101 production medium (D medium plus 0.15% soybean oil, vol / vol).

D medium

Dextrin (CPC modified starch) 1 40.0 g

Stillage 7.0 g

Yeast extract 5.0 g

C0CI2 • 6H2O 50.0 mg distilled water 1000 ml before sterilization, the pH is adjusted to 7.3 with HaOH

The fermentation reactors are operated at 24 ° C using a stirring rate of 640 rpm (approximately Kd 5.5) and an air flow of 0.5 WM for 72 h. Defoamer, "Hodag" -MF (Hodag Chemical Corp.) is used as needed, but not more than 0.1%.

The content of the two fermentation containers is combined after the fermentation attempt; you get about 201.

200 ml portions of the batch are centrifuged in a “Servali” RC-2B centrifuge at 9000 rpm for 20 min. The protruding material is filtered through a 1 cm layer of «Hyflo Super-Cel» in a 33.02 cm Lapp funnel with a filter cloth. The filtrate has a bioactivity of 20 units / ml.

The filtered broth (181) is applied to a column (8.2 x 29 cm) made of "Dowex" -l x 2 (C1 ~), 0.297 to 0.149 mm, at 150 ml / min. The column is then washed with 11 deionized water and then with 1510.15 M NaCl + 0.01 M tris-HCl buffer, pH 7.0 + 25 (in neutral EDTA in 50% MeOH at 150 ml / min .

The antibiotic 890Aio is then neutral with 3.2% NaCl + 0.02 M tris-HCl buffer, pH 7.0 + 25 jxM. EDTA in 80% MeOH eluted at 100 ml / min. The following fractions are collected: a fraction of 11, four fractions of 500 ml each and twelve fractions of 11 each. The bioactivity and the absorption at 220 nm are determined by each fraction, and such fractions which have bioactivity / A22o ratios above 0, 6 units / A22o unit and which contain more than 10% of the total activity / fraction obtained are combined for further purification. Fractions 4 to 8 are thus combined, which contain a total of 22% of the bioactivity used.

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10th

The collected fractions are concentrated to 190 ml under reduced pressure. The precipitated salt is washed with deionized water and the wash water is added to the supernatant. This brings the total volume to 220 ml. The pH of the concentrate is adjusted to 6.5 with HCl and the eluate is applied to a column (6.0x59 cm) made of “Amberlite” XAD-2, which was previously mixed with 8160% aqueous acetone and then with 161 deionized water was washed. After application is complete, the column is rinsed with 5 x 10 ml portions of deionized water and the antibiotics are eluted with deionized water at 35 ml / min. The following fractions are collected: a fraction of 500 ml, then seven fractions of each 250 ml, then four 500 ml fractions. I5

The bioactivity and HAEA304 values of fractions 3 to 12 are measured. Fractions 4 to 9 have HAEA304 / A22o ratios above 0.01 and are combined for further purification. Fraction 3, which contains about Vs of the applied salt, is also added to this combined material 20. The combined material contains 469 HAEA304 units.

The combined fractions are diluted to 4.11 and they are applied to a column (2.15 x 42 cm) made of "Dowex" -l x4 (Cl ~), (-0.037 mm) at 2 ml / min. The column is washed with 100 25 ml of 50% methanol and the antibiotic is washed with 0.25 M NaCl + 0.01 M NH4C1 + 0.0001 M NH3 in 80%

MeOH eluted at about 2 ml / min. Fractions from 2 to 12 ml are collected.

Bioactivity and absorption at 220 nm, 260 nm and 30 300 nm are determined for every fifth fraction and HAEA300 is measured for the bioactive peak fractions. The 890Aio bioactivity occurs in fractions 85 to 150 with a maximum in fraction 120. Fractions with HAEA3oo / A3oo values above 0.05 are pooled and 35 further purified. Fractions 105 to 133, which contain a total of 130 HAEA3oo units, are combined in this way. The combined fractions 105 to 133 are diluted to 1900 ml with deionized water and the pH is adjusted to 7.6. The sample is applied to a column (2.15 x 42 cm) on "Dowex" -l x2 40 (Cl ~), (-0.037 nm), previously with 313% NaCl in 50% methanol, followed by 50 ml 50% methanol was washed. After application of the entire sample, the column is washed with 100 ml of 30% methanol and eluted at 2 ml / min with 0.26 M NaCl + 0.005 M NH4Cl + 0.0002 M NH3 in 45 30% methanol. Fractions of 10 to 12 ml are collected.

The main peak of the 890Aio antibiotic occurs in fractions 230 to 290 with a maximum in fraction 260. The fractions with an A300 / A250 ratio above 1.10 50 and an A3oo / A22o ratio above 0.83 are collected for further purification. Fractions 240 to 275 are thus combined, which contain 91.6 HAEAsoo units.

The combined fractions 240 to 275 are concentrated to 10 ml under reduced pressure and the concentrate is separated from the precipitated salt 55 by pipetting. The salt is washed with 2 ml of deionized water and the wash water is added to the concentrate. The combined 12 ml concentrate and wash water are applied to a column (2.15X76cm) of "Bio-Gel" P-2 (0.074 to 0.037mm), previously with 30 ml saturated NaCl in deionized water, followed by 1500 ml of deionized water and 50 ml of 0.05 mmol NH3 in deionized water. After the application has ended, the column is rinsed with 3 × 1 ml of 0.05 mM NH3 in deionized water. The antibiotic is eluted with 0.05 mM NH3 in deionized water at 0.73 ml / min. Fractions of 3,651 each are collected.

The main peak of the 890Aio antibiotic occurs in fractions 29 to 50 with a maximum in fraction 36. The fractions with an A3oo / A25o ratio above 1.9 and also with an A3oo / A22o ratio above 1.45 are combined for lyophilization and NMR analysis. Fractions 36 to 45 are thus combined which contain 50.8 A3oo units, 45.1 of which can be extinguished by hydroxylamine.

The combined fractions 36 to 45 are concentrated to 1 ml under reduced pressure and 5 ml of D2O are added. The sample is again concentrated to 0.835 ml and 0.825 ml of it is transferred to a glass ampoule, freeze-dried and lyophilized. The lyophilized sample contains 48.2 A3oo units and 4.6 mg solids. It is referred to as sample 9441-154.

The minor fractions 31 to 34 and 46 to 49 are combined; a «side sample» is obtained, which is then concentrated to 1 ml, quickly frozen and lyophilized. These fractions contain 24 A3oo units, 17 of which can be deleted by hydroxylamine. This sample is referred to as Sample 9441-155.

Example 3

A frozen ampoule containing 2 ml MA-4638 inoculum is slowly thawed and the contents are transferred aseptically into a vaccination flask containing 500 ml of C medium. The inoculation flask, 21, a shake flask with triple deflection elements, which is equipped with its side arm, is closed with cotton or cotton wool.

C-medium autolyzed yeast («Ardamine») * 10.0 g

Glucose 10.0g

MgS04 • 7H2O 0.05 g

Phosphate buffer ** 2.0 ml distilled water 1000 ml before sterilization, the pH is adjusted to 6.5 with NaOH * "Ardamine": Yeast Products, Inc.

** Phosphate buffer solution:

KH2PO4 91.0g

NaîHP04 95.0 g distilled water 1000 ml

The inoculated inoculation flask is shaken for 36 hours at 28 ° C + 1 ° C from a 210 rpm gyrotation shaker, 5.08 cm vibration amplitude.

The growth from this inoculation flask is used to inoculate a 141 glass fermentation container containing 101 production medium (D medium plus 0.15% soybean oil, vol / vol).

D medium

Dextrin (CPC modified starch) 40.0 g

Stillage 7.0 g

Yeast extract 5.0 g

C0CI2 • 6H2O 50.0 mg distilled water 1000 ml before sterilization, the pH is adjusted to 7.3 with NaOH

The fermentation tank is operated at 24 ° C using a stirring rate of 550 rpm (Kd 3.0 to 3.5) and an air flow of 0.5 WM. Defoamer, "Hodag" -MF (Hodag Chemical Corp.) may be used but not more than 0.1%.

The culture broth is harvested after 72 hours of fermentation.

11

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200 ml portions of the batch are centrifuged on a «Servali» RC-2 centrifuge at 9000 rpm. The supernatant contains 71 with 39 bioassay units / ml.

The excess material is filtered through a Lapp funnel, which contains a 1 cm layer of «Hyflo Super-Cel» on a filter cloth. 3.25 ml of 0.1 M neutral EDTA are added to 6.51 filtrate, and the filtrate is then applied to a column (8.2 × 21.5 cm) of “Dowex” -lx 2 (C1 ~), ( 0.297 to 0.149 mm), applied at 60 ml / min. After use, the column is rinsed with 11 deionized water and then washed with 1210.15 M NaCl + 0.02 M tris-HCl buffer, pH 7.08, + 25 (jM EDTA in 50% MeOH at 50 ml / min .,

The antibiotic 890Aio is then eluted with 12.313% NaCl + 0.02 M tris-HCl buffer, pH 7.1, + 25 p, M EDTA in 80% MeOH at 60 ml / min.

The following fractions are collected: a fraction of 500 ml, four fractions of 200 ml each, twelve fractions of 500 ml each and five fractions of 11 each. Bioactivity occurs in fractions 3 to 17 with a maximum in fractions 6,7 and 8 on. The fractions with bioactivity / A22o values above 0.9 units / A22o unit, which also contain more than 8% of the total recovered activity / fraction, are collected for further purification. Fractions 6 to 12 are thus combined, which contain 106,000 bioassay units or 41% of the bioactivity used. These combined fractions are kept at -60 ° C. until they are combined with fractions 6 to 13 from a second Dowex column, as described in Example 4.

Example 4

Two frozen ampoules, each containing 2 ml MA-4638 inoculum, are slowly thawed. The contents are transferred aseptically into two inoculation flasks, each containing 500 ml of C medium. The inoculation flasks, 21 shake flasks with triple deflecting elements and a side arm, are closed with cotton or cotton wool.

C-medium autolyzed yeast («Ardamine») * 10.0 g

Glucose 10.0g

MgS04-7H20 0.05 g

Phosphate buffer ** 2.0 ml distilled water 1000 ml before sterilization, the pH is adjusted to 6.5 with NaOH * "Ardamine": Yeast Products, Inc.

** Phosphate buffer solution:

KH2PO4 91.0 g

NazHPOt 95.0 g distilled water 1000 ml

The inoculated inoculation flasks are shaken for 36 hours at 28 ° ± 1 ° C on a 210 rpm gyrotation shaker, 5.08 cm vibration amplitude.

The growth from the inoculation flasks is used to inoculate two 141 glass fermentation containers, each containing 101 D production medium.

D medium

Dextrin (CPC modified starch) 40.0 g

Stillage 7.0 g

Yeast extract 5.0 g

C0CI2 • 6H2O 50.0 mg distilled water 1000 ml before sterilization, the pH is adjusted to 7.3 with NaOH

Two fermentation vessels are loaded and operated at 24 ° C using a stirring rate of 550 rpm (Kd 3.0 to 3.5) and an air flow of 0.5 WM. If desired, a defoaming agent, Polyglykol s 2000, is used, but not more than 0.1%.

The culture broths from the two fermentation containers are harvested after 72 hours.

200 ml portions of the 10-1 batches are centrifuged in a «Servall» centrifuge at 9000 rpm. 91 supernatant material is obtained from each batch. The bioanalyses show that the batch contains 123.4 units / ml and the batch II 31.8 units / ml.

The batches are combined, cooled and filtered through a 1 cm layer of "Hyflo Super-Cel" on a 33.02 cm "LabTek" -15 porcelain book funnel. 7.5 ml of 0.1 M neutral EDTA are added to the 151 filtrate.

The filtrate is applied to a column (13.4 x 16 cm) made of "Dowex" -l x2 (Cl ~), (0.297 to 0.149 mm) at 100 ml / min. After completion of the application, the column is washed with 21 20 deionized water and then with 2010.15 M NaCl + 0.01 M tris-HCl buffer, pH 7.01, + 25 pM EDTA in 50% MeOH with 40 to 80 ml / min washed.

The antibiotic 890Aió is then eluted with 24.41 of a solution containing 3% NaCl + 0.02 M tris-HCl, pH 7.0, + 25 | xM EDTA 25 in 80% MeOH, at 100 ml / min . The following fractions are collected: one fraction of 800 ml, four fractions of 400 ml each, twelve fractions of 1000 ml each and five fractions of 2000 ml each.

Antibiotic activity occurs in fractions 5 to 16 30 with a maximum in fractions 7 and 8. The total activity in these fractions is 212,000 units, corresponding to 51% of the activity used. Fractions 6 to 13 are pooled for further purification.

The pooled fractions 6 to 13 from Dowex column 35 are added to pooled fractions 6 to 12 from the Dowex column of Example 3 and concentrated to 2.61 under reduced pressure in a long tube, steam jacket evaporator. This concentrate is further reduced to 40,300 ml by evaporation under reduced pressure in a rotary evaporator, and the precipitated salt crystals are filtered off.

The pH of the concentrate is adjusted to 6.48 by adding 1.7 ml of concentrated hydrochloric acid. The concentrate is then placed on a column (8.1 x 59 cm) of 45 "Amberlite" XAD-2 and the column is rinsed with 3x20 ml batches of deionized water. The antibiotic is eluted with deionized water at a flow rate of 60 ml / min. The following fractions are collected: a fraction of 11, seven fractions of 500 ml each and eight fractions of 1000 ml each. These are cooled in ice immediately after collection.

The bioanalysis and HAEA3oo values as well as the A220, A260 and Ä3oo values for each fraction are determined. The fractions with HAEA3oo / A3oo values above 0.075 are pooled 55 for further purification. Fractions 5 to 9 containing 164,000 bioassay units are combined.

The collected fractions are concentrated to 100 ml under reduced pressure and 200 ml of methanol are added. The sample is then applied to a column (2.15 x 38 cm) 60 made of "Dowex" -l x4 (Cl "), (-0.037 mm) at 2 ml / min.

After use, the column is washed with 100 ml of 80% methanol and the antibiotic is eluted with 0.26 M NaCl + 0.005 M NH4C1 + 0.00005 M NH3 in 80% MeOH at 2 ml / min. Fractions of ie 10 ml are collected 65.

The bioactivities and the values for A300, A250, A220 and HAEA300 are determined by every fifth fraction. The fractions with HAEA3oo / A2oo ratios above 0.07 will be

634104

12

25th

30th

united for further cleaning. Fractions 125 to 150 are combined in this way.

The lyophilized sample 9441-155 from Example 2, redissolved in 1.0 ml of deionized water, is added to the combined fractions 125 to 150 from the “Dowex” 1 × 4 column. This combined sample is diluted to 1900 ml with deionized water and applied to a «Dowex» -l x 2 (C1 ~) column (2.15 x 40 cm), (-0.037 mm) at 2 ml / min. The column is rinsed with 150 ml of 30% methanol. The antibiotic is then eluted with 610.25 M NaCl + 0.005 M NH4CI + 0.00005 M 10 NH3 in 30% MeOH at 2 ml / min. Fractions of 11.1 ml each are collected.

The absorption at 300 nm, 250 nm and 220 nm is measured by every fifth fraction. The main peak of the antibiotic 890Aio occurs in fractions 360 to 430 with a '5 maximum in fractions 394 to 400. All fractions with an A3oo / A22o ratio above 1.30 are pooled for further purification. Fractions 380 to 420 are thus combined. These pooled fractions contain 202 A3oo units, 197 of which can be erased by hydroxylamine. 20th

Sample 9441-154 from Example 2, which was dissolved in 1 ml of D2O plus 4 ml of deionized water and contains 30.5 HAEA3oo units, is added to the combined fractions 380 to 420. The combined sample is diluted to 2.01 with deionized water and placed on a column (2.15 x41 cm) made of «Dowex» -lx 2 (Cl ~), (0.074 to 0.037 mm), with 0.6 to 2 ml / min applied. The column is rinsed with 360 ml of 30% methanol. The antibiotic 890Aio is eluted with 0.25 M NaCl + 0.005 M NH4CI + 0.00005 M NH3 in 30% MeOH at 1.9 ml / min. Fractions of 9.7 ml are collected.

The absorption at 300 nm, 250 nm and 220 nm is determined by every fifth fraction. The main peak of the antibiotic 890Aio, determined by the absorption at 300 nm, occurs in the fractions 270 to 358 with a maximum in the fraction 315. The fractions with A3oo / A22o ratios 35 over 1.75 are collected for further purification. Fractions 295 to 335 are thus combined, which contain 145 A3oo units, of which 137 can be extinguished by hydroxylamine.

The combined fractions 295 to 335 are concentrated to 10 ml under reduced pressure. The concentrate is adjusted to pH 7.0 with 1 M NaOH. The concentrate is applied to a column (2.15 x 75 cm) made of "Bio-Gel" P-2 (0.074 to 0.037 mm) and the column is rinsed with 3 x 1 ml batches of deionized water. The antibiotic is eluted with deionized water at 0.6 ml / min. 6.4 ml fractions are collected.

The absorption values at 300 nm, 250 nm and 220 nm as well as the conductivities of every second fraction are determined. The main absorption peak at 300 nm occurs in the 50 fractions 9 to 27 with a maximum in the fraction 14. Fractions 11 through 16 have conductivities proportional to the A3oo value and these fractions are pooled for mass spectrometric and NMR analysis. This combined material contains 75.4 A3oo units in 38.7 55 ml. Of which 34.7 ml are concentrated to 1.22 ml. A 1.20 ml portion of it is frozen and lyophilized for NMR analysis. 2.84 mg of solids are obtained. The remaining 4 ml are transferred to the NH4 + form by passing through a column (0.7 x 15 cm) made of «Dowex» -50W x 2 (NH4 +), (0.074 to 0.037 mm). The resulting ammonium salt is concentrated to 0.11 ml, 10 (xl are removed, 100 ml of deionized water are added, and four 40 ml samples are removed and individually frozen and lyophilized for mass spectrometric analysis [9441-209-2] . 65

Biogel fractions 17 to 23, which contain 29.4 A3oo units, are also concentrated to 1 ml, frozen and lyophilized; 1.80 mg of solids are obtained.

Example 5

Two frozen ampoules, each containing 1 ml of MA-4638 culture broth, are slowly thawed and the contents are aseptically transferred to two 250 ml Erlenmeyer flasks with triple deflecting elements, each containing 50 ml of C inoculation medium. The flasks are closed with cotton.

C-medium autolyzed yeast («Ardamine») * 10.0 g

Glucose 10.0g

MgS04-7H20 0.05 g

Phosphate buffer ** 2.0 ml distilled water 1000 ml before treatment in the autoclave, the pH is adjusted to 6.5 with NaOH * "Ardamine": Yeast Products, Inc.

** Phosphate buffer solution:

KH2PO4 91.0 g

Na2HP04 95.0 g distilled water 1000 ml

The two inoculation flasks are shaken for 20 to 24 hours at 28 ° + 1 ° C on a 210 rpm gyrotation device, 5.08 cm vibration amplitude. The contents are then collected and used to inoculate the production flasks.

Ten 21 shake flasks with deflection elements, each containing 300 ml D-production medium, are inoculated with 10 ml / flask with the broth from the inoculation flask. The above production flasks are closed with gauze.

D medium

Dextrin (CPC modified starch) 40.0 g stillage 7.0 g yeast extract 5.0 g

C0CI2 • 6H2O 50.0 mg distilled water 1000 ml before treatment in the autoclave, the pH is adjusted to 7.3 with NaOH

After the inoculation, the production flasks are at

Incubate 24 ± 1 ° C for 3 days with shaking on a 210 rpm gyrotation device, 5.08 cm vibration amplitude. The flasks are harvested, the contents are collected and the broth is examined for its activity.

Harvest age (h) 72

pH 6.5

890 assay (units / ml) 30.45

21 of the entire broth from the above D production flasks are centrifuged in 200 ml parts at 9000 rpm; 1.71 supernatant material with a pH of 6.8 is obtained.

The supernatant material is applied to a column made of "Dowex" -lx 2 (CI "), (0.297 to 0.149 mm), layer dimensions 3.9 cm x 25 cm, at a flow rate of 5 to 10 ml / min. The column is with 50 ml deionized water, followed by 21 0.15 M NaCl + 0.02 M tris-HCl buffer, pH 7.0 +

25 (xM neutral EDTA in 50% (vol / vol) aqueous methanol.

The product is then treated with 30 g / 1 NaCl + 0.02 M tris-HCl buffer, pH 7.0, + 25 | IM EDTA in 80% (vol / vol) aqueous methanol at a flow rate of 5 ml / min eluted. Fractions of 10 ml are collected.

Bioactivity occurs in fractions 5 to 180 with a maximum in fractions 25 to 70. Fractions 12-105 are pooled and concentrated to 15 ml under reduced pressure. The pH is adjusted to 6.5 with 1 M HCl.

13

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The concentrate is applied to a column (3.4 x 55 cm) made of XAD-2, which was washed with 2.5160% (vol / vol) aqueous acetion, deionized water and 5% NaCl in deionized water . The column is rinsed with 3x5 ml portions of deionized water and eluted with deionized water at 10 ml / min. Fractions of 10 ml are collected.

Bioactivity occurs in fractions 32 to 270 with a maximum in fractions 42 to 62. The total bioactivity that is eluted 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. All of the pooled fractions are concentrated to 50 ml under reduced pressure and contain 120 HAEA304 units.

The concentrate is diluted with 200 ml of methanol and applied at 2 ml / min to a column (2.15x40 cm) made of «Dowex» -l x4, (-0.037 mm), which was previously washed with 21 of 30 g / 1 NaCl in 80% aqueous methanol and 1180% aqueous methanol. After application of the sample, the column is washed with 100 ml of 80% (vol / vol) aqueous methanol and with 2.510.22 M NaCl + 0.01 M NH4CI + 0.0002 M NH3 in 80% (vol / vol) aqueous Methanol followed by 310.31 M NaCl + 0.01 M NH4CI + 0.0002 M NHs in 80% (vol / vol) aqueous methanol. The flow rate is 2 ml / min. Fractions of 10 ml each are collected.

The antibiotic 890Aio is separated on the «Dowex» -l x4 column, eluted in fractions 155 to 195, determined by analysis on ATCC 8461.

Antibiotic 890Ai

A tube of lyophilized culture from Streptomyces flavogriseus MA-4600 NRRL 8140 is opened aseptically and the contents are suspended in a tube containing 1.5 ml of sterile medium E of the following composition.

Medium E

Yeast extract 10.0g

Glucose 10.0g

MgS04-7H20 0.05 g

Phosphate buffer ** 2.0 ml distilled water 1000 ml ** Phosphate buffer solution:

KH2PO4 91.0g

NazHPCh 95.0 g distilled water 1000 ml

This suspension is used to inoculate a 250 ml Erlen-meyer inoculation flask with triple deflection elements, which contains 54 ml of inoculation medium C of the following composition.

Medium C

autolyzed yeast 10.0 g («Ardamine») *

Glucose 10.0g

MgS04-7H20 0.05 g

Phosphate buffer ** 2.0 ml distilled water 1000 ml the pH is adjusted to 6.5 with NaOH * "Ardamine": Yeast Products Corporation

** Phosphate buffer solution:

KH2PO4 91.0 g

N32HPÛ4 95.0 g distilled water 1000 ml

The inoculation flask is closed with cotton or cotton wool and shaken for 30 hours at 28 ° ± 1 ° C on a 220 rpm gyrotation shaker, 5.08 cm vibration amplitude.

Fifty 250 ml Erlenmeyer production flasks without deflection elements, each containing 40 ml of production medium F, are inoculated with 1 ml / flask of the broth from the inoculation flask. The production flasks are closed with cotton.

Medium F

Tomato paste or pulp 20.0 g primary yeast 10.0 g

Dextrin («Amidex») 20.0 g

CoCl2-6H20 5.0 mg distilled water 1000 ml the pH is adjusted to 7.2 to 7.4 with NaOH.

After inoculation, the production flasks are incubated for 3 days at 28 ° ± 1 ° C with shaking on a 220 rpm gyrotation shaker, 5.08 cm vibration amplitude. The flasks are tested for activity against standard Vibrio percolans ATCC 8461 analytical discs using 1.27 cm analytical discs immersed in samples of the centrifuged fermentation broth. The samples are diluted with 0.05 M phosphate buffer, pH 7.4. The results are summarized in the following table.

Harvest age (h) 72

pH 6.4 Vibrio percolans

('/ 100 dilution) Assay 23 mm

890 assay, units / ml 103

The entire broth is centrifuged in 200 ml parts in polycarbonate bottles for 15 min at 9000 rpm; 1600 ml of combined supernatant material with a potency of 104 units / ml are obtained. 0.5 ml 0.1 M neutral EDTA is added.

The centrifuged broth is adsorbed on a column of «Dowex» -l x 2 (Cl ~), (0.297 to 0.149 mm), layer dimensions 3.8 x 22 cm, at a flow rate of 6 to 20 ml / min. The column is rinsed with 100 ml of deionized water and with 11 deionized water containing 50 g of sodium chloride, 0.02 M tris-HCl buffer, pH 7.0 and 25 n, M neutral EDTA at a flow rate of 6 ml / min eluted. Fractions of 10 ml are collected.

The antibiotic 890Ai occurs in fractions 13 to 81 with a maximum in fractions 25 to 33 if one counts from the start of the first application of the salt eluate. Fractions 24 to 41 with the highest biopotency / a220 ratios are pooled for further treatment. The combined fractions contain a total of 29,000 units or 17% of the bioactivity applied.

The «Dowex» eluate is concentrated to 10 ml, the pH is adjusted to 6.5 with dilute hydrochloric acid and the concentrate is applied to a column of XAD-2, layer dimensions 3.3x36 cm, previously with 2160 % aqueous acetone, deionized water and 5% (w / v) sodium chloride in deionized water. The sample is eluted with deionized water at a flow rate of 6 ml / min. Fractions from 40 to 260 ml are collected.

Antibiotic activity occurs in fractions 6 to 14, which range from 220 to 2560 ml of eluted volume. The peak occurs at fractions 9 and 10, which differ from 370

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

634104

14

extend to 590 ml eluted volume. Fractions 9 to 12, which range from 370 to 1060 ml of eluted volume, have the highest HAEAaoo / Azzo ratios and are combined for further treatment. These fractions have 36,600 units and this corresponds to 126% of the apparent activity used.

The combined fractions 9 to 12 are concentrated to 100 ml, and the concentrate is applied at a flow rate of 2 ml / min onto a column of «Dowex» -l x4 (Cl ~), (-0.037 mm), layer dimensions 2.2x41 cm , applied. The column is rinsed with 50 ml deionized water and eluted with 310.07 M NaCl + 0.005 M NH4CI + 0.0001 M NH3 in deionized water at a rate of 2 ml / min. 10.8 ml fractions are collected starting from the first application of the eluent.

The main peak of antibiotic 890Ai occurs in fractions 181 to 217 with a maximum in fraction 198. Fractions 186 to 210, which contain a total of 114 absorption units at 300 nm, are collected.

The collected fractions are concentrated to 4.0 ml and the pH is adjusted to 7.3 by adding 16 ml of 1 M NaOH. The concentrate is applied to a column of "Bio-Gel" P-2, (0.0074 to 0.031 mm), layer dimension 5 2.15 x 70 cm, and washed with 3 x 1 ml portions of deionized water and with deionized water 0.96 ml / min eluted. 3.85 ml fractions are collected.

The main peak of antibiotic 890Ai occurs in fractions 24 to 44 with a maximum in fractions 33 and 10 34. Fractions 27 to 38 with the highest A3oo / A245 ratios are pooled for lyophilization. These combined fractions have a total of 72 A3oo 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 adding 10 μl of 0.1 M NaOH. The sample is divided into two parts of 1.50 ml each, and the parts are quickly frozen separately and lyophilized from 14 ml ampoules with a glass screw cap, each sample contains 1.73 mg 890Ai, this corresponds to 35.8 A3oo units.

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