CH636127A5 - Process for the preparation of the novel antibiotic MSD890A9 - Google Patents

Description

The present invention relates to a method for producing the new antibiotic MSD 890A9, which is active against both gram-positive and gram-negative bacteria. The antibiotic is made by growing or growing species of Streptomyces on suitable fermentation media.

The discovery of the remarkable antibiotic properties of penicillin has stimulated a great deal of interest in this area. As a result, many other valuable antibiotic substances were found. In general, the antibacterial activity of each of these antibiotics does not include certain clinically important pathogenic bacteria. For example, some are mainly only active against gram-positive types of bacteria. Resistance to the widespread use of existing antibiotics in the treatment of bacterial infections has caused a serious resistance problem to arise.

The disadvantages of the known antibiotics have therefore stimulated further research into other antibiotics which are active against a large range of pathogens and also against resistant strains of particular microorganisms.

The invention comprises a method for producing the antibiotic MSD 890A9, in particular in dilute form, as a raw concentrate or in pure form.

The present invention has for its object to provide a method for producing a new and useful antibiotic which is highly effective in inhibiting the growth of various gram-negative and gram-positive microorganisms.

A method for the production of the new antibiotic compound by fermentation of a nutrient medium with species of Streptomyces is to be created.

The new antibiotic compound produced by the process according to the invention is produced by growth under controlled conditions of a new strain of Streptomyces flavogriseus.

Based on extensive taxonomic research, the strain of the microorganism used in the present invention was identified as belonging to the species Streptomyces flavogriseus and was designated MA-4638 15 in the depository of Merck & Co., Inc. Rahway, N.J. His culture became permanent on September 15, 1976 with no restrictions on availability at the Culture Collection of the Northern Regional Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, 20th Peoria, III. It is available under accession number 11 020.

Streptomyces flavogriseus MA-4638 forms the antibiotic 890As, which is isolated from the fermentation broth in essentially pure form.

25 The morphological and cultural properties of Streptomyces flavogriseus MA-4638 are listed in the following table.

Morphology - spore carriers branched, straight to curved spore chains that form tufts. The chains are • 30 no longer than 10 spores. The spores are spherical to oval -0.9 p-x 1.2 [i (970x).

Culture properties of oatmeal agar (ISP Medium 3)

35 Vegetative growth - reverse yellow-dark yellow lined with brown, ruffled;

Aerial mycelium - light gray lined with medium gray;

soluble pigment - none;

Czapek Dox Agar (Sucrose Nitrate Agar)

40 vegetative growth - reverse brown lined with dark brown;

Aerial mycelium - medium gray, velvety;

soluble pigment - slight browning of the medium; Egg Albagar 45 vegetative growth - reverse-yellow-dark yellow lined with brown;

Aerial mycelium - medium gray, mixed with yellowish gray (2dc) and gray yellow (2db);

soluble pigment - light yellowish dark yellow; so glycerin-asparagine agar vegetative growth - reverse brown;

Aerial mycelium - velvety, light gray with yellowish tone (2dc);

soluble pigment - light dark yellow;

55 Inorganic Salt Starch Agar (ISP Medium 4)

vegetative growth - reversed-greenish-yellowish-dark-yellow;

Aerial mycelium - velvety, medium gray with yellow tone (3fe); soluble pigment - very light dark yellow; 60 yeast extract-dextrose + salts-agar vegetative growth - reverse-dark brown; Aerial mycelium - dark gray, mixed with lighter gray; soluble pigment - none;

Yeast extract malt extract agar (ISP Medium 2)

65 vegetative growth - reverse brown;

Aerial mycelium - velvety, dark gray lined with lighter gray;

soluble pigment - none;

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Skim milk agar vegetative growth - dark yellow;

Aerial mycelium - scanty, whitish;

soluble pigment - slight 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; Become alkaline;

Nutrient tyrosine agar vegetative growth - reverse-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;

HzS formation - negative;

Nutrient agar vegetative growth - reverse-light grayish brown;

Aerial mycelium - light gray lined with dark gray;

soluble pigment - none;

Agile 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;

Slice of potato 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;

Vegetable growth - dark yellow;

Aerial mycelium - none;

soluble pigment - none;

Liquefaction of gelatin - completely.

All of the above determinations were 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 Edition (1958), Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus MA-4638 will continue to be tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganisms are grown on synthetic base medium (Pridham and Gottlieb), which contains 1% carbohydrate, at 28 ° C. for three weeks. The pH of the media used in these tests is approximately neutral (6.8 to 7.2). Table I lists the usability of these carbohydrate sources by Streptomyces flavogriseus MA-4638; + means growth; ± means bad or questionable growth; and - means no growth compared to a negative control (no carbon source).

Table I

glucose

+

Maltose

+

Arabinose

+

Mannitol

+

Cellulose

-

Mannose

-L

Fructose

+

Raffinose

-

Inositol

-

Rhamnose

+

Lactose

+

Sucrose

±

Xylose

+

The strength of growth with changes in temperature 25 and the oxygen requirement of the microorganism are as follows: temperature range (yeast extract dextrose + salt agar) 28 ° C. - good vegetative growth and air growth; 37 ° C - good vegetative growth; no aerial hyphae; 50 ° C - no growth.

30 Oxygen requirement (sting culture in yeast extract dextrose + salt agar)

aerobic.

The method according to the invention is not limited to the use of the organism Streptomyces flavogriseus or to organisms which completely meet the above growth and the microscopic properties. These are listed for illustration only. The use of mutants which are obtained from the organisms described by various means or methods, such as X-ray radiation, ultraviolet radiation, nitrogen mustard, exposure to bacteriophages and the like, is desired and intended.

890As is formed during aerobic fermentation under controlled conditions from suitable, aqueous nutrient media that are inoculated with a strain of the organism Streptomyces 45 flavogriseus. Aqueous media, such as those used to make other antibiotics,

are suitable for the production of 890As. Such media contain sources of carbon, nitrogen and inorganic salts which are assimilable by the microorganisms. 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, e.g. of oats, rye, corn starch, corn flour and the like can be used, either alone or together with a source of assimilable carbon 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 multiple such carbon sources can be combined 60 in the medium. In general, many proteinaceous materials can be used as nitrogen sources in the fermentation process. Suitable nitrogen sources include, for example, yeast hydrolysates, primary yeast, soybean meal, cottonseed meal, hydrolysates of casein, corn steep liquor, soluble substances which are produced in the production of brandy, or stillage or the like. The preferred source are soluble substances from the distillery or stillage. The nitrogen sources are either alone or in a mixture

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4th

preferably used in amounts in the range from about 0.2 to 6% by weight, based on the aqueous medium.

The inorganic nutrient salts which are added to the culture media include the commonly used salts which give sodium, potassium, ammonium, calcium, magnesium, phosphate, sulfate, chloride, carbonate and similar ions. In addition, 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 conveniently carried out at a temperature in the range from 20 to 37 ° C. For optimal results, however, it is preferred to carry out the fermentation at a temperature of 23 to 28 ° C. The initial pH of the nutrient medium used for growing the strains of Streptomyces flavogriseus cultures and producing antibiotic 890As can vary from about 6.0 to 8.0.

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

The fermentation can be initiated in a sterilized flask of the nutrient medium through one or more stages of inoculum development. The nutrient medium used for the inoculation stage can be any suitable mixture of carbon and nitrogen sources. The inoculation flask is shaken in a constant temperature chamber at about 28 ° C for one day or until growth is sufficient, and part of the resulting growth is used to inoculate either a second stage inoculum or the production medium. Intermediate stage seeding flasks, when used, are developed in essentially the same way, 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 towards the end of the incubation period the contents of the flask are centrifuged or filtered.

For work 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 the tank and sterilized by heating at a temperature up to about 120 ° C. After cooling, the sterilized medium is inoculated with a previously grown inoculum of the producing culture, and the fermentation can proceed for a period of, for example, 1 to 6 days while the nutrient medium is agitated and / or aerated and the temperature at about 22 holds up to 26 ° C. This method of producing the 890A9 antibiotic is particularly suitable for producing large quantities of antibiotics.

Physical and chemical properties of the 890As antibiotic

Antibiotic 890A9 is an acidic compound found in RichCH3-CH

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 migrates 8.0 cm in 30 min, compared to a 4.0 5 cm migration for 890Ai. The disodium salt is a white or slightly yellow powder, lyophilized from an aqueous solution. In acidic conditions in aqueous solution, the antibiotic in the free acid form is unstable. The antibiotic is therefore usually found in a bound form as a salt or other derivative that is more stable.

The disodium salt of the antibiotic 890As has an absorption maximum at 308 and 228 nm and a minimum at 262 nm at neutral pH in water. For the most purified preparation, the A3os / A26o ratio is 2.05; the A308 / A220 ratio 1.17; and the A3os / A228 ratio is 1.05. More than 93% of the absorption at 308 nm is extinguished by reaction with hydroxylamine at neutral pH. After the reaction with hydroxylamine, the absorption increases at 260 nm and the ratio of the increase at 20 260 nm to the decrease at 308 nm is approximately 0.30. The reaction with hydroxylamine, which follows the A3os decrease under the conditions described in the section "Hydroxylamine Reaction", is obviously first order with a half-life at room temperature of 25 to 50 s. 25 Determined against a standard of 890Ai antibiotic, the 890A9 antibiotic has 82 units / HAEA3os unit. HAEA308 is described in the section “Hydroxylamine reaction”.

Table II lists the 100 MHz signals of the nuclear magnetic resonance spectrum of 890A9 in D2O at 10 ° C. Chemical shifts are given in ppm, based on HOD, at 4.70 5 and 32 ° C, and the coupling constants are given in Hertz.

35

Table II

CHsCH 1.55 (3H, D, 6Hz)

CH3CO 2.12 (3H, S)

Ce-H 3.91 (1H, D, D; J6-5 = 5.5 Hz; J6.s = 9 Hz)

40 Cs-H 4.36 (1H, M)

C8-H ~ 5 (partially overlaid by the HOD line)

C (1) -H2 3.14 (1H, D, D; 18.2 + 9.8 Hz)

-S-CH = CH-N 6.10 (1H, D, 13.9 Hz) and 7.19 (1H, D, 13.9 Hz)

45

The mass spectrum of TMSÌ-890A9 is characterized by the fragments listed in Table III.

Table III

m / e

55

84 227 298/9

339

340 366 456

Antibiotic 890A9 has the following molecular structure

5

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

Trying to determine the antibiotic spectrum profiles of the 890A antibiotic? is carried out by applying a 0.015 ml droplet from a 20 μg / ml aqueous solution of the antibiotic to the surface of a 100 × 15 mm petri dish which contains 5 ml inoculated nutrient agar plus 0.2% yeast extract and which is incubated at 25 ° C. The results, expressed as the diameter in mm of the inhibition zone or zone, are shown in Table IV.

Organism zone of inhibition

Diameter, 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 ACC 8461 32

Xanthomonas vesicatoria MB No. 815 28

Proteus vulgaris ATCC 21100 35

Escherichia coli MB No. 1418 30

Pseudomonas stutzeri AT CC 11607 10

Klebsiella pneumoniae MB No. 1264 24

Aerobacter aerogenes MB No. 835 25

Erwinia atroseptica ATCC 4446 30

Pseudomonas aeruginosa MB No. 2824 0

Corynebacterium pseudodiph. ATCC 9742 19

Escherichia coli ATCC 9637 26

Streptococcus faecium MB No. 2820 10

Streptococcus agalactiae MB No. 2875 29

Vibrio percolans MB No. 2566 (res. Ceph C) 21

Proteus vulgaris MB No. 2112 (Episom) 34

Proteus mirabilis MB No. 3126 27

Vibrio percolans ATCC 8461 + 2x 105 | i / ml 19 penicillinase

Vibrio percolans ATCC 6461 + β-lactamase 32 from Enterobacter clacae MB 2646

Antibiotic 890As is effective against various gram-positive and gram-negative bacteria and is a potent inhibitor of bacterial ß-lactamases and can be used in human and veterinary medicine. 890A9 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. Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae and Salmonella schottmuelleri.

The described compound can also be used together with β-lactam antibiotics, which are sensitive to β-lactamases, to potentiate the action of such β-lactam antibiotics by inhibiting the lactamase activity and thus enables an extended lifespan of the antibiotics.

A mixture of antibiotic 890Ai with a lactamase-sensitive β-lactam antibiotic becomes more effective in the treatment of infections with β-lactamase-producing

Bacteria are considered to be the same amount of ß-lactamase sensitive antibiotics alone.

Antibiotic 890Aç can be used as an additive to animal feed, for the preservation of feed and food and as a disinfectant. It can be found in aqueous formulations in amounts ranging from about 0.1 to about 100 parts antibiotic / million parts solution or preferably in concentrations ranging from about 1 to about 10 parts antibiotics / million parts solution to destroy and inhibit the growth of harmful bacteria medical and dental objects or facilities and used as a bactericide in industrial applications.

Antibiotic 890A9 can be used in pharmaceutical preparations as the only active ingredient or together with one or more 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 methods known to those skilled in the art or that can be adapted to the antibiotic described.

The compositions described may contain, in addition to a carrier, other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, viscosity regulators or flavoring agents, agents which are well known and commonly used.

In veterinary medicine, such as in the treatment of poultry, cows or cattle, sheep, pigs and the like, the preparations can e.g. can be used as an intramammary preparation in either long-acting or rapidly releasing raw materials.

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

In the treatment of bacterial infections in humans or living beings, the compounds described can be administered orally or parenterally together with a β-lactamase-sensitive antibiotic by methods known per se for the administration of antibiotics. The 890A9 antibiotic can be administered in an amount of about 2 to 600 mg / kg / day. It is preferred in amounts of about 5 to 100 mg / kg / day, preferably in divided dose units, e.g. three to four times a day. It can be administered in dosage units, e.g. Contain 100,330,400 or 1000 mg of active ingredient with suitable physiologically acceptable carriers or diluents. The dose units will be in the form of liquid preparations, such as solutions or suspensions, or as solids in tablets or capsules. The optimal dose in a given case will depend on the type and severity of the infection to be treated. Smaller doses will be used in pediatrics; all of these settings are 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 890As, e.g. the pharmacologically acceptable salts formed with inorganic and organic bases, for example the 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 which from primary, secondary or tertiary amines, such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkali

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

636 127

6

nolamines, lower-alkylenediamines, N, N-diaralkyl-lower-alkylenediamines, aralkylamines, amino-substituted-lower-alkanols, N, N-di-lower-alkylamino-substituted-lower-alkanols, amino-polyamino- and guanidino-subst. lower alkanoic acids and nitrogen-containing heterocyclic amines. 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, procain, ethanolamine, morphine, benzylamine, ethylenediamine, N, N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-l-propanol, theophylline, N-methylgluca -min etc. deduce.

The salts of the described compound can be isolated directly from the fermentation medium using suitable eluents during the ion exchange chromatography or they can be prepared by methods 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 I mol of product (I) plus I 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). Salts can also be prepared by treating 1 mole of the product with 1 mole of a base with a divalent cation. The salts produced by the process according to the invention are pharmacologically acceptable, non-toxic derivatives which can be used as an active ingredient in a suitable pharmaceutical dosage unit form. They can also be combined with other drugs to produce preparations with a wide range of activities.

The fermentation broths containing the antibiotic 890A9 produced according to the invention generally have activities in the range from about 2 to 170 units / ml when analyzed according to the disk diffusion assay using Vibrio percolans (ATCC 8461). The antibiotic 890A contained in these fermentation broths? can be recovered and purified using a number of methods. In one of these methods, the 890As antibiotic can be adsorbed on a strongly basic anion exchange resin. Examples of such strongly basic anion exchange resins are those containing a styrene-divinylbenzene matrix, e.g. the polystyrene resin "Dowex" 1 x2 with quaternary ammonium groups on the core (manufactured by Dow Chemical Co., Midland, Michigan), in the chloride cycle. Other examples of these classes are strong base exchange resins including 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, Philadelphia 5, Pennsylvania).

The adsorbed antibiotic can be easily eluted from the anion exchange resin with brine in 80% (vol / vol) aqueous methanol. The eluate obtained in this way can be further purified by other cleaning methods. The eluate can be purified by concentrating it and passing it through a column filled with a polystyrene, non-polar, hydrophobic, cross-linked divinylbenzene polymer such as XAD-1, 2 and 4, or 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, Washington Square, Philadelphia, Pennsylvania).

A method of making further purified 890A9 antibiotic can be performed, for example, by gel filtration with 5 polyacrylamide gel having a pore size excluding molecules with a molecular weight above 1800, such as "Bio-Gel" P-2 (manufactured by Bio-Rd, Richmond, California) will. Other gels, such as "Sephadex" G-10, can also be used for desalination.

The preferred method according to which the 890A9 antibiotic can be obtained in high purity from a broth is by centrifuging or filtering the broth to remove the solids, the filtrate on an anion exchange resin such as "Dowex" -l x2 in the chloride cycle adsorbed with 15 3% NaCl in 80% (vol / vol) aqueous methanol and eluted. As a result, the antibiotics are both concentrated and partially cleaned. It is then passed over a column with suitably prepared XAD-2, which delays the antibiotics and thereby cleans and desalinates the «Dowex» -1 x2 eluate 20. The fractions enriched with 890A9 are collected and further purified. Chromatography on a «Dowex» -l x2, particle size max. 0.037 mm, with elution by NaCl and / or NH4CI in 80% aqueous methanol gives a product that is free of most UV 25 absorbing impurities (the NH4CI is used to maintain a certain buffer capacity in the eluent). The 890A9 is separated from other antibiotics in this procedure. By desalting "Bio-Gel" P-2 or "Sephadex" G-10 in 50% methanol, the majority of the salt that was introduced in the "Dowex" -1 x2 chromatography is removed.

The remaining impurities can be reduced by a further chromatography cycle on "Dowex" -1 x2 of the specified particle size and elution with a solution containing sodium chloride and 50% methanol, followed by desalination.

When purifying by column chromatography, generally only those fractions of eluted volume which contain antibiotic in an amount which is at least 30% pure are combined as purest fractions for further purification. The criteria for cleaning are the ratios bioactivity / A220 or A308 / A260 and HAEA308 / A220 and the conductivity in desalination processes. At each chromatography level, A220, A260, A308 and the bioactivity of suitable fractions are measured. If possible, HAEA308 is also measured and the conductivity is measured during desalting. The criteria for deciding which fractions to combine for the subsequent processes can be adjusted somewhat so that one obtains a higher yield at the expense of purity or vice versa, so that one obtains a higher purity at the expense of the yield.

When working on a laboratory scale (less than 201 sample volumes), all chromatography steps, with the exception of XAD-2 chromatography, can be carried out in a cold room at 2 to 55 5 ° C. XAD-2 chromatography is carried out at room temperature. The pH of the antibiotic solutions to be stored is adjusted to 7 to 8 by carefully adding dilute NaOH or NCl solutions. Aqueous solutions are stored in a refrigerator or, preferably before -60, in ice-water, and 50-80% methanol solutions are stored at -20 to -60 ° C.

For purification steps prior to XAD-2 chromatography, the solutions are generally adjusted to 25 µM in EDTA by adding a volume of a solution of 0.1M Na2 65 EDTA, which is neutral by adding sodium hydroxide (0.1 M) EDTA ») was adjusted to a pH of 7.0.

7

636 127

A flow chart for the cleaning process to make the 890A9 antibiotic is shown below.

the cleaning process for

Antibiotic 890An v

.1. Cooling

2. Centrifuge

3. Addition from EDTA

Centrifuged broth

In

1. Adsorbed on Dowex-1x2

(Cl-) 0.297-0.149 mm

2. Wash with 0.15M NaCl + 0.01M tris-HCl + 25 / UM EDTA in 50% MeOH '

3. Wash with water

4. Elute with 3% NaCl + 0.01M tris-HCl, pH 7, + 0.25 / uM neutral.

, EDTA in 80% methanol

"Dowex-1x2

1. Concentrate

2. Adsorb to XAD-2 ^ 3. Elute with water

XAD-2!

Eluate

1. Concentrated fractions containing 890An

2. Dilute with MeOH

3. Adsorb. on "Dowex-1 x4 (Cl ~) (-0.037 mm)

4.Eluting with 0.26M NaCl + 0.005M NHaCI + O, 00005M NH *

! r | In 80 # methanol

) DowexMx4

Eluate 890A ^

1.Dilute with water

2.Adsorb.an 'Dowex-1x2 (Cl ~) (-0.037 mm)

3.Eluting with 0.30M NaCl + 0.005M NHaCI + 0.001M NH *

H_ ^ 1 ig-. 5090 methanol

"DowexMx2 eluate

1. Concentrate

2. Add 1 volume of MeOH

3. Adsorb. to "Bio-Gel8 P-2 (in 50% MeOH)

4. Elute with 0.2mM NH, in 50% MeOH D

890 Ag

'Sio-Gel * P-2

Eluate

1. Concentrate

2. Lyophilize

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8th

Concentration (| j.g / ml)

Zone diameter (mm)

3.12

16.8

6.25

22.3

12.5

25.0

25th

29.6

Assay procedure for antibiotic 890A9 II. Analysis or assay procedure for the determination of

I. Bioassay «890 assay units»

An agar plate diffusion process is described under an agar plate diffusion method known per se.

Use of Vibrio percolans ATCC 8461 as a test organism using Vibrio percolans ATCC

mus used. A purified sample of the antibiotic 890Ai 5 8461 was used as the test organism. Cephaloridine is used as the standard. Antibiotic 890Ai is used according to the standard. Vibrio percolans ATCC 8461 containing prepared according to the method described below. Panels are made as follows. A culture of

Plates containing Vibrio percolans ATCC 8461 become Vibrio percolans ATCC 8461 in a nutrient broth.

manufactured as follows. Yeast extract overnight on a rotary shaker

A lyophilized culture of Vibrio percolans ATCC 8461 ion is incubated at 28 ° C and then diluted to a density of 15 ml in sterilized medium which is 8 g / 1 "Difco" nutrient-60% permeability at 660 nm. A 33.2 ml portion of

broth and 2 g / 1 yeast extract in distilled water, ser diluted culture is added to 1 1 medium, the one

"Nutrient broth yeast extract" (hereinafter referred to as NB YE contains nutrient agar plus 0.2% yeast extract and is kept at 46 ° C), suspended. The culture is rotated overnight. The inoculated medium containing agar is incubated on an ion shaker at 28 ° C. This culture is poured into 100 x 15 mm plastic petri dishes, 10 ml per dish.

for inoculating the surface of slant cultures that are 1.5% cooled and at 2 to 4 ° C up to 5 days before use

Agar contained in NBYE used. The inoculated slant kept.

cultures are incubated overnight at 28 ° C and then in The concentration of cephaloridine, which is equivalent to 1

stored in a refrigerator. Unit / ml 890Ai is determined by analysis on plates that like

The chilled slant cultures made from a single lyo 20 described above but containing 5 ml inocu-

philized culture, contain up to 4 weeks of gelled medium / plate, determined as follows,

used from its manufacture as follows: An eyelet Four concentrations of cephaloridine are used as the standard of inoculum from the slant culture in 50 ml NBYE, which is used in - 3.12, 6.25, 12.5 and 25 µg / ml with 12 .5 (ig / ml is contained as a 250 ml Erlenmeyer flask. The comparative solution. The zone diameter on a 5 ml plate of culture overnight on a rotary shaker 25 for the standard are as follows:

incubated at 28 ° C and then diluted to a density that is 50%

Permeability at 660 nm results. A 33.2 ml portion of this diluted culture is added to 11 NBYE containing 15 g agar and kept at 46 ° C. The inoculated medium containing agar is placed in 100x 15 mm plastic petri dishes,

5 ml per bowl, poured, chilled and kept at 2 to 4 ° C up to 5 days before use.

Filter paper disks with a diameter of 1.27 cm are immersed in the solution to be examined and defined as one unit as the amount of antibiotic / ml

laid the agar. Alternatively, the disks can be pinned to give a 25 mm zone of inhibition on a 5 ml plate as described in petting V10 ml solution loaded on the dry disk part I above. Therefore, in this experiment, and then the disc can be placed on the agar, a concentration of 12.5 (ig / ml cephaloridine equivalent to that. The diameter of the zone of inhibition is assumed to be 1 unit 890Ai / ml measured the slope of the line incubation (12 to 24 h at 25 ° C.) Given - for cephaloridine is 4.0, the calculations are made of the dilutions of the solutions, if any, that are examined using a slope of 4.0

should be carried out with 0.05 M potassium phosphate buffer, pH 7.4 «potassium.

umphosphate buffer »(hereinafter referred to as KPB) or with deionized water. III. Hydroxylamine reaction

The efficacies or potencies are calculated as follows - Antibiotic 890A9 reacts with hydroxylamine and gives a calculated result. An inclination is determined by taking 45 a substance in which the absorption at 308 nm is essentially

Zone diameter of a solution of the antibiotic 890A9 and Chen has disappeared. This provides a basis for a four-fold dilution (in KPB) of this solution. a quantitative analysis of the 890A9 antibiotic.

The solution to be analyzed is analyzed for 0.05 M in potassium gene plate, and the average zone size phosphate, pH 7.4 by adding V20 vol. Of a solution, which is determined at each concentration . The slope is equal to 50 0.8 M K2HPO4 and 0.2 M KH2PO4. ! / 100 vol. of half the difference in the average zone size of 1 M hydroxylamine hydrochloride is added and these sen. The efficacies or potencies are calculated after the For absorption at 308 nm in intervals of Vi to 2 min mei: measured. The implementation is carried out at room temperature

. \ guided. First order kinetic values are assumed

Potency (units / ml) = (^) 55 men and the half-life is derived from the decrease in absorption

Inclination) during the first 10 min. From this half-life

(Potency of the standard) x dilution x 10 the time is estimated at which no further absorption absorption

where D is the average diameter that should be observed from the discomfort, and the observations will

Known formed zones, Ds the average through over this point in time. If one measures the standard zones and «dilution» the degree, with 6o no further decrease over this time, the one that the unknown was diluted before analysis, means the total absorption decrease as «hydroxylamine-vanishing. If no standard is used, it is assumed that Ds bare absorption at 308 nm (HAEA3os) is taken (whereby for 0.25 mm and the (potency of the standard) is taken as 1 in the dilution effect and the absorption of the hydroxyl unit / ml assumed, determined on Vibrio percolans ATCC mins is corrected). Taking an 8461 absorption decrease. Pure 890 Ai is defined to observe an efficacy 65 over this time, the rate of background decrease from 250 units per hydroxylamine-disappearing absorption decrease is calculated, and the observed decrease to absorption unit at 300 nm results if it is used as standard this time is corrected for the background decrease

becomes. greed, assuming that the background decrease with

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time is linear. The corrected value is then recorded as HAEA308.

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

The following examples explain the processes by which the products described are manufactured. The manufacture of the 890Ai antibiotic is described in US application Ser. 634,300. Expressis verbis is referred to this publication.

example 1

A slant culture containing MA-4638 is used to inoculate a 250 ml Erlenmeyer flask equipped with baffles and containing 50 ml Medium A.

Medium A

Dextrose 10.0g

Yeast autolysate («Ardamine» *) 10.0 g

MgS04-7H20 0.05 g

Phosphate buffer ** 2.0 ml distilled water 1000 ml pH 6.5

* "Ardamine": Yeast Products, Inc. ** Phosphate buffer solution:

KH2PO4 91.0g

NazHPOt 95.0 g distilled water 1000 ml

This flask is shaken at 28 ° C on a 220 rpm shaker with a vibration amplitude of 5.08 cm for 2 days until the growth is satisfactory. After 2 days, this inoculum is used to inoculate three 250 ml Erlenmeyer flasks containing 40 ml Medium B

using 2 ml / flask (5%).

Medium B

Tomato paste or marrow 20.0 g

Whole wheat (ground) 20.0 g distilled water 1000 ml pH: adjusted to 7.0 using NaOH.

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. After 3 days of age, an aliquot of the supernatant solution from the centrifuged broth is used for classification and identification testing. Using 0.63 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. The bioautography of an electrophoretogram of the centrifuged broth shows two components. The faster moving smaller patch contains 890As.

Example 2

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

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

Glucose 10.0g

MgSÛ4 * 7H20 0.05 g

Phosphate buffer ** 2.0 ml distilled water 1000 ml the pH is adjusted to 6.5 with NaOH before treatment in the autoclave

* "Ardamine": Yeast Products, Inc.

** Phosphate buffer solution:

KH2PO4 91.0g

Na2HP04 95.0 g distilled. Water 1000 ml

The inoculation flasks are shaken for 20 to 24 hours at 28 ° C ± 1 ° C on a 210 rpm gyrotation shaker, 5.08 cm (2 inch) vibration amplitude. The broth from the inoculation flasks is used to inoculate production flasks.

The following different production flasks containing D production medium are used: six 21 shake flasks without deflection elements, which contain 150 ml medium / flask, sealed with cotton; eight 21 shake flasks with three deflection elements, which contain 350 ml medium / flask, with gauze closure; and two hundred and fifty 250 ml shake flasks without baffles containing 40 ml medium / flask sealed with cotton. The levels of inoculum used are: 5 ml / 150 ml medium; 10 ml / 350 ml medium; and 1.5 ml / 40 ml medium.

D medium

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

CoC12-6H20 50.0 mg distilled water 1000 ml the pH is adjusted to 7.3 with NaOH before the treatment in the autoclave

After the inoculation, the production flasks are shaken at 25 ° ± 1 ° C with shaking on a 220 rpm gyrotation shaker, 5.08 cm vibration amplitude, for 3 days. The flasks are harvested and the contents are combined.

The broth is centrifuged in 200 ml portions at 11,000 rpm for 15 minutes. To the combined supernatant solutions, a total of 91, 2 ml of 0.1 M EDTA are added and the supernatant solution is applied to a column (8.2 x 20 cm) of Dowex-1 x 2 (Cl ~), 0.297 to 0.149 mm applied, which was previously washed with 510.1 M HCl + 30 g / 1 NaCl in 80% (vol / vol) aqueous methanol and then with 51 deionized water. The speed of application is 10 to 50 ml / min. After application of the sample, the column becomes neutral with 11 deionized water and then with 9 10.15 M NaCl + 0.01 M tris-HCl buffer, pH 7.0, + 25 | iM. EDTA washed in 50% aqueous methanol. The antibiotic 890A9 is then treated with 91% of 30 g / 1 NaCl ± 0.01 M tris-HCl buffer, pH 7.0, + 25 μm in neutral EDTA in 80% aqueous methanol at a flow rate of 40 ml / min eluted. The fractions from 200 to 900 ml are collected.

The antibiotic activity according to the ATCC 8461 analysis appears to occur in all fractions 1 to 19, with a broad maximum in fractions 6 to 11 ranging from 1290 to 3550 ml of eluted volume. Fractions 6 to 14 are pooled and concentrated to 190 ml under reduced pressure and the pH is brought from 7.6 to 6.5 by adding 0.4 ml of 12M HCl.

The concentrate is applied to a column (4.95 x 36 cm) made of XAD-2, which was previously washed with 41% of 60% aqueous acetone, deionized water and 50 g / 1 NaCl in deionized water. After application of the sample, the column is washed three times with 101 deionized water and then eluted with deionized water at 15 ml / min. The fractions from 100 to 500 ml are collected. This column is washed and the chromatography is carried out at room temperature. The diluted fractions are immediately cooled with ice water.

The antibiotic activity according to the ATCC 8461 analysis

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

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10th

occurs in fractions 4 to 12, which range from 450 to 2345 ml of eluted volume. Fractions 5 to 8, which range from 600 to 1245 ml eluted volume, have the highest ratios of HAEA304 / A220 and are combined accordingly for further purification. The 5 fractions collected have a total of 607 HAEA304 units.

The combined fractions 5 to 8 are concentrated to 40 ml under reduced pressure and the concentrate is diluted with 160 ml of methanol. The pH of this solution is adjusted to 7.5 with 1 M NaOH, and the solution is then placed on a column, 2.2 x 40 cm, made of "Dowex" -l x4 (Cl ~), (-0.037 mm) , which was previously washed with 210.1 M HCl + 30 g / 1 NaCl in 80% (vol / vol) aqueous methanol and then with 1180% methanol. The rate of application of the sample is 2 ml / min. After use, column 15 is washed with 50 ml of 80% methanol and eluted with 0.26 M NaCl + 0.01 M NH4Cl + 0.0002 M NH3 in 80% methanol at 1 ml / min. The 10 ml fractions are collected.

The bioactivity according to the ATCC 8461 analysis occurs in fractions 87 to 280. The main peak of the antibiotic 20 890A? occurs in fractions 231 to 275, the maximum activity occurs in fraction 250. Fractions 231 to 275 are pooled to give the 890As "Dowex" batch.

The 890A <> - "Dowex" batch (combined fractions 231 to 275) is diluted with 1600 ml of deionized water and a column (2.2 x 42 cm) made of "Dowex" -l x2 (Cl ~) (- 0.037 mm), applied at 2 ml / min. After use, the column is washed with 50 ml of 50% (vol / vol) methanol and with 3.4 10.30 M NaCl + 0.010 M NH4CI + 0.0002 M NHs in 50% methanol and then with 210.30 M NaCl + 0.01 M 30 NH4CI ± 0.0002 M NH3 in 60% methanol at 2 ml / min. 9.5 ml fractions are collected.

The main absorption peak at 305 nm occurs in fractions 385 to 440 with a maximum in fraction 410. Fractions 400 to 425 have the highest A3os / A26o ratios and are combined for further processing. The combined fractions contain 160 A305 units, of which 147 can be deleted by hydroxylamine.

The combined fractions are concentrated to 5.5 ml under reduced pressure. The pH is determined by 40

Add 30 μl of 1M NaOH to 7.1, and 5.5 ml of methanol are added. The sample is then applied to a column (2.2 x 69 cm) made of "Sephadex" G-10, which has been washed beforehand with 210.05 mM NH3 in 50% (vol / vol) methanol. After application of the sample, the column is eluted with 0.05 45 mM NH3 in 50% methanol at 1 ml / min. 2.95 ml fractions are collected.

The main absorption peak at 307 nm occurs in fractions 69 to 85 with a maximum in fractions 77 to 78. Conductivity measurements of individual fractions show that 50 of the main absorption peak at 307 nm elutes somewhat after the salt peak. Fractions 74 to 82 containing 68 A307 units with a ratio of A307 / A260 of 1.80 are pooled.

The pooled fractions are concentrated to 3 ml under reduced pressure and the concentrate is applied to a column (3.4 x 45 cm) of "Dowex" -50x2 (Na +) (0.074 to 0.037 mm), previously with 2110 ~ 5 M NaOH in deionized water and then washed with 100 ml deionized water. After application of the sample, the 60 column is rinsed twice with 2 ml of deionized water and then eluted with deionized water at 4 ml / min. 3.95 ml fractions are collected.

The main absorption peak at 307 nm occurs in fractions 38 to 48 with a maximum in fractions 42 and 65 43. Fractions 42, 43 and 44 have the highest A307 / A22o ratios and are collected for lyophilization. The pH of the pooled fractions is determined by

Add 2.2 jo.10.1 M NaOH from 5.8 to 7.0 and remove 2.5 ml. The remaining 9.4 ml are concentrated to 1 ml under reduced pressure and 10 ml of D2O are added. The concentration to 1 ml and the addition of 10 ml of D2O are repeated and the final concentration is reached. 1 ml of the concentrate is frozen and lyophilized. 20 mg of solids are obtained, of which about 18 mg are NaCl, estimated by the conductivity.

Example 3

Two frozen ampoules, each containing 1 ml of MA-4638 culture broth, are slowly thawed and the contents are aseptically placed in two 250 ml Erlenmeyer flasks with three baffles, each containing 50 ml of C inoculation medium. The flasks are closed with cotton.

C-medium autolyzed yeast («Ardamine» *)

glucose

MgS04-7H20

Phosphate buffer **

distilled water

10.0 g 10.0 g 0.05 g 2.0 ml 1000 ml The pH is adjusted to 6.5 by adding NaOH before the treatment in the autoclave.

* "Ardamine": Yeast Products, Inc.

** Phosphate buffer solution:

KH2PO4 91.0 g

Na2HP04 95.0 g distilled. Water 1000 ml

The inoculation flasks are shaken for 20 to 24 hours at 28 ° ± 1 ° C on a 210 rpm gyrotation shaker, 5.08 cm vibration amplitude. The content is collected and used to inoculate the production flask.

Ten 21 shake flasks with deflection elements, each containing 300 ml of D production medium, are inoculated with 10 ml / flask of the broth from the inoculation flask. The production flasks are covered with a gauze closure.

D medium

Dextrin (CPC modified starch)

Stillage

Yeast extract

CoCh-óHìO

distilled water

40.0 g 7.0 g 5.0 g 50.0 mg 1000 ml the pH is adjusted to 7.3 with NaOH before the treatment in the autoclave

After inoculation, the production flasks are incubated at 24 ° + 1 ° C with shaking on a 210 rpm gyrotation shaker, 5.08 cm vibration amplitude, for 3 days. The flasks are harvested, the contents are combined and the broth is checked for activity.

Harvest age (h) 72

pH 6.5

890 assay (units / ml) 30.45

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

The supernatant solution is applied to a «Dowex» 1x2 (Cl -) column, 50 to 100 mesh (0.297 to 0.149 mm), layer dimension 3.9 cm x 25 cm, at a flow rate of 5 to 10 ml / min. The column is washed with 50 ml of deionized water and then with 210.15 M NaCl + 0.02 M tris-HCl buffer, pH 7.0, + 0.25 µM neutral EDTA in 50% (vol / vol) aqueous Washed methanol.

The product is then washed with 30 g / 1 NaCl + 0.02 M tris-HCl buffer, pH 7.0, + 25 µM EDTA in 80% (vol / vol) aqueous solution.

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like to elute methanol at a flow rate of 5 ml / min. Fractions of 10 ml are collected.

The bioactivity occurs in fractions 5 to 180 with a maximum in fractions 25 to 70. Fractions 12-105 are combined and concentrated to 15-5 ml under reduced pressure. The pH is adjusted to 6.5 with 1 M HCl and the concentrate is applied to a column (3.4 x 55 cm) made of XAD-2, which is 2.51 of 60% (vol / vol) aqueous acetone, deionized water and 5% NaCl in deionized water. The column is rinsed with 3 x 10 ml portions of deionized water and eluted with deionized water at 10 ml / min. Fractions of 10 ml are collected.

The 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 pooled and the stored pooled fractions 12 to 34 from the XAD-2 column of Example 1 are added. All of the combined fractions are concentrated to 50 ml under reduced pressure: 2o they contain 120 HAEA304 units.

The concentrate is diluted with 200 ml of methanol and applied at 2 ml / min to a column (2.15 x 40 cm) made of «Dowex» -l x4, (-0.037 mm), previously with 21 of 30 g / 1 NaCl in 80% aqueous methanol and 1180% aqueous methanol was washed. 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 and then eluted with 310.31 M NaCl + 0.01 M NH4CI + 0.0002 M 30 NH3 in 80% (vol / vol) aqueous methanol. The flow rate is 2 ml / min. Fractions of 10 ml each are collected.

The antibiotic 890As is separated on the «Dowex» -1 x4 column and eluted in fractions 280 to 350, determined 35 by analysis on ATCC 8461.

The "Dowex" -l x4 fractions 307 to 338 are combined and concentrated to 7.6 ml under reduced pressure. 30 ml of 1 M NaOH are added to adjust the pH to 7.0 to 7.5. The concentrate is diluted with 7.6 ml of methanol and the solution is centrifuged to remove the salt deposit. The supernatant solution is concentrated to 4 ml under reduced pressure, and 6 ml of methanol are added to this concentrate. The salt precipitate can settle and the supernatant solution is pipetted onto a column 45 (2.2 x 70 cm) made of «Sephadex» G-10, which has been previously treated with 10 ml 1 M NH3 in aqueous methanol and then with 11 0.0001 M. NH3 was washed 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 washed with 400 ml of the same solvent at a flow rate of 1 ml / min. The column is then eluted with 0.00005 M NH3 in 50% aqueous methanol at a flow rate of 1 ml / min. 10 ml fractions are collected,

counting from the start of the first application of the 50% 55 methanol eluate.

An absorption peak at 305 nm occurs in fractions 9 to 22 with a maximum in fraction 12. Fractions 11 to 17 are pooled; they contain 17 A305 units, of which 9.1 can be deleted by hydroxylamine. eo

The combined fractions are concentrated to 2 ml under reduced pressure, diluted to 10 ml with 99.7% D2O and then concentrated to 1.5 ml under reduced pressure, frozen and lyophilized. The resulting white powder, which contains the product 890A9 and more than 5 mg sodium chloride 65, is analyzed by NMR spectroscopy.

Antibiotic 890Ai

A tube or ampoule of lyophilized culture of Streptomyces glavogriseus MA-4600 NRRL 8140 is opened aseptically and the contents are suspended in a tube which contains 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 ml distilled water 1000 ml

** Phosphate buffer:

KH2PO4 91.0g

Na2HP04 95.0 g distilled water 1000 ml

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

10.0 g 10.0 g 0.05 g 2 ml 1000 ml

Medium C

autolyzed yeast («Ardamine» *)

Glucose MgS04-7H20 phosphate buffer **

distilled water the pH is adjusted to 6.5 with NaOH * «Ardamine»:

Yeast Products, Corp.

** Phosphate buffer solution:

KH2PO4 91.0g

Na2HP04 95.0 g distilled. Water 1000 ml

The inoculation flask is closed with cotton or cotton wool and shaken for 30 h 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 wool or cotton.

Medium F

Tomato paste or pulp primary yeast dextrin («Amidex») C0CI2.6H2O distilled water

20.0 g 10.0 g 20.0 g 5.0 mg 1000 ml the pH is adjusted to 7.2 to 7.4 with NaOH

After inoculation, the production flasks are shaken at 28 ° ± 1 ° C with shaking on a 220 rpm gyrotation shaker, 5.08 cm vibration amplitude, for 3 days. The flasks are assayed for activity against Vibrio percolan's ATCC 8461 standard assay plates using 1.27 cm analysis disks immersed in centrifuged fermentation broth samples. The samples are diluted with 0.05 M phosphate buffer, pH 7.4. The results are summarized in the following table.

Hours after removal 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; you get 1600

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12

ml combined supernatant solution with a potency of 104 units / ml. 0.5 ml 0.1 M neutral EDTA is added.

The centrifuged broth is adsorbed on a «Dowex» -1 x2 (Cl ~), (0.297 to 0.149 mm) column, layer dimensions 3.8 x 22 cm, at a flow rate of 6 to 20 ml / min. The column is rinsed with 100 ml deionized water and eluted with 11 deionized water containing 50 g sodium chloride, 0.02 M tris-HCl buffer, pH 7.0 and 25 nM neutral EDTA at a flow rate of 6 ml / min . Fractions of 10 ml are collected. Antibiotic 890Ai occurs in fractions 13 to 81 with a maximum in fractions 25 to 33 when counting from the start of the first application of the salt eluate. Fractions 24 to 41, which have the highest biopotency / A22o ratios, are pooled for further treatment. The combined fractions have a total of 29,000 units or 17% of the bioactivity used.

The «Dowex» eluate is concentrated to 10 ml and the pH is adjusted to 6.5 with dilute hydrochloric acid. The concentrate is applied to a column of XAD-2, layer dimensions 3.3 x 36 cm, which was previously washed with 2 160% acetone, deionized water and 5% (w / v) sodium chloride in deionized water . The sample is then eluted with deionized water at a flow rate of 6 ml / min. The 40 to 260 ml fractions are collected.

Antibiotic activity occurs in fractions 6 to 14, which range from 220 to 2560 ml of the eluted volume. The peak occurs at fractions 9 and 10, which range from 370 to 590 ml of eluted volume. Fractions 9 to 12, which correspond to 370 to 1060 ml of the eluted volume, have the highest HAEA3oo / A22o ratios and are combined for further treatment. These fractions contain 36,600 units; this corresponds to 126% of the obviously used activity.

The combined fractions 9 to 12 are concentrated to 100 ml and the concentrate is applied to a column of “Dowex” -l × 4 (Cl ~) (−0.037 mm), layer dimension 2.2 × 41 cm, at a flow rate of 2 ml / min . The column is rinsed with 50 ml of deionized water and eluted with 31 0.07 M NaCl + 0.005 M NHiCl + 0.0001 M NHs in deionized water at a flow rate of 2 ml / min. Fractions of 10.8 ml are collected starting with the first application of the eluent.

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

The combined fractions are concentrated to 4.0 ml, 15 and the pH is adjusted to 7.3 by adding 16 ^ .11 M NaOH. The concentrate is applied to a column of "Bio-Gel" P-2 (0.074 to 0.037 mm), layer dimension 2.15 x 70 cm, washed with 3 x 1 ml deionized water and with deionized water at 0.96 ml / min eluted. 3.85 ml fractions are collected.

The main peak of the 890Ai antibiotic occurs in fractions 24 to 44 with a maximum in fractions 33 and 34. Fractions 27 to 38, which have the highest A300 / A245 ratios, are pooled 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 30 7.5 by adding 10 ml of 0.1 M NaOH. The sample is divided into two parts of 1 , 50 ml divided 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, which corresponds to 35.8 A3oo units.

20th

25th

G

Claims (2)

636 127 PATENT CLAIMS 1. Process for the preparation of the new antibiotic MSD 890A9 in the form of the compound of the formula OSO..H I 3 CH -CH Ì S-CH = CH-NH-C-CH. CO OH or of their pharmaceutically acceptable salts, characterized in that a strain of Streptomyces flavogriseus providing 890A9 is cultivated in aqueous nutrient medium which contains assimilable sources of carbon, nitrogen and inorganic salts, under submerged, aerobic conditions and the antibiotic is obtained. 2. The method according to claim 1, characterized in that the cultivated microorganism is Streptomyces flavogriseus NRRL11 020.