BE511759A - - Google Patents

Description

       

   <Desc / Clms Page number 1>
 



  IMPROVEMENTS RELATING TO CHEMICAL COMPOSITIONS AND PROCESSES
OF PREPARATION OF THESE COMPOSITIONS.



   The present invention relates to processes for the preparation of novel compounds exhibiting antibiotic properties.



   The present invention relates to a new process for obtaining an improved antibiotic composition consisting of erythromycin and its acid addition salts.



   The present invention also relates to a process for the preparation of erythromycin which consists in cultivating a variety of Streptomyces erythreus producing erythromycin in a culture broth containing assimilable sources of carbohydrate, nitrogen and inorganic salts. until appreciable antibiotic activity is produced by this organism in the culture broth, and the erythromycin is recovered from this culture broth.



   -As regards the preparation of the new compounds which are the subject of the present invention, this relates to the preparation of a nitrogenous base which is arbitrarily called “erythromycin” and of the acid addition salts of this base. These include a compound which for convenience will be referred to herein as an "erythromycin complex" an acid addition salt comprising erythromycin and an unidentified organic acid.



   Erythromycin and its acid addition salts are characterized by a broad antibacterial spectrum. They have antibiotic activity against many microorganisms, which take or do not take the gram.



  Another important antibiotic property of the compounds is their ability to inhibit the growth and development of some of the Rickettsial-like bodies and large viruses, for example epidemic typhus and meningo-pneumonitis, and to effectively inhibit the growth. and the development of certain spirocheteso The antibiotic properties of the compounds as well as their low toxicities make them of great utility as therapeutic agents in the treatment of numerous diseases
The antibiotic activity of erythromycin against organisms

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 representative mes is given in Table Sa. Antibiotic activity was determined either by tests known in the United States of America under the name of

    "streak-dilution" and "broth-dilution". In the first test, the test organisms were spread out on a series of agar plates containing varying concentrations of the antibiotic to determine the minimum erythromycin concentration in meg. (micrograms) per cm3 of substrate that inhibited growth for a period of 40 hours. In the last test, the test organisms grew in a nutrient broth.
 EMI2.1
 tif containing varying amounts of erythrom, ycin.

   The presence of about 10% horse serum in the broth has no noticeable effect on the antibacterial activity of the antibiotics. In the table, the letters (ag) and (bd) follow the concentration. Inhibitory treatment correspond to the test methods (agar-agar dilution test and culture broth dilution test, respectively) which were used. Erythromycin salts possess antibiotic activities corresponding to the values given in the table, taking into account the increased molecular weights of the salts.



   TABLE I.
 EMI2.2
 
<tb>



  Test <SEP> organism <SEP> Inhibitory <SEP> concentration
<tb>
<tb> ¯¯¯¯ <SEP> mcg / cm3
<tb>
 
 EMI2.3
 Microcoocus p-vogenes var. aureus 0, $ (ag)
 EMI2.4
 
<tb> Micrococcus <SEP> pyogenes <SEP> var. <SEP> aureus <SEP> 0.4 <SEP> (<SEP> ag) <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> (resistant <SEP> to <SEP> penicillin <SEP>)
<tb>
 
 EMI2.5
 Microcossus pvpgenes var. avreus 0.8 (ag) Micrococcus 1 op: enes var * aureus 0.8 (ag)
 EMI2.6
 
<tb> (resistant <SEP> to <SEP> streptomycin <SEP>)
<tb>
 
 EMI2.7
 Listeria monogztogenes 0.31 (bd)
 EMI2.8
 
<tb> Bacillus <SEP> subtilis <SEP> 0.2 <SEP> (ag)
<tb> Vibrio <SEP> cholera <SEP> 0.63 <SEP> (bd)
<tb>
 
 EMI2.9
 Rvcobacterium sp.

   (607) 6.2 (ag) Rvcrobacterium phlei 0.8 (ag) Hemophilus¯, pertussis. 0.31 (bd) Mycobacterium avium 1.6 (ag) Mvcobagteriwu smeronatis 6.2 ¯ (ag) Klebsiella pneumoniae 6.2 (ag)
 EMI2.10
 
<tb> Brucella <SEP> melitensis <SEP> 1.56 <SEP> (bd)
<tb>
<tb> Brucella <SEP> am <SEP> 1.56 <SEP> (bd)
<tb>
<tb> Neisseria <SEP> intracellularis <SEP> 5.0 <SEP> (bd)
<tb>
 
 EMI2.11
 Diploegoeus PDeumoniB.! 3 0.02 (bd)
 EMI2.12
 
<tb> (Variety <SEP> sensitive <SEP> to <SEP> sulfonamides)
<tb>
<tb> Streptococcus <SEP> pyogenes <SEP> 0.02 <SEP> (bd)
<tb>
<tb> Hemophilus <SEP> influenzae, <SEP> 1.25 <SEP> (bd)
<tb>
<tb> Streptococcus <SEP> viridans <SEP> 0.16 <SEP> (bd)
<tb>
 
 EMI2.13
 02 = ebacterium dip, hteriae 0.02 (bd)
 EMI2.14
 
<tb> Vibrio <SEP> comma <SEP> 6.25 <SEP> (bd)
<tb>
 
As regards the new processes which are the subject of the present invention,

   this encompasses fermentation processes comprising the
 EMI2.15
 growth of an actinomvcete in a culture broth containing available sources of carbohydrate, nitrogen and mineral salts.

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 EMI3.1
 



  The actynomyoete used in the fermentation processes which are the subject of the present invention belongs to the genus Streptomyces of the order of Acetinvçetales, in accordance with the classification established in the work f'Bergev's Manual'of Determinative Bacteriology "(6th Edition) , page 938.



  By its morphological characteristics, the organism appears to be closely related to an actinomycete described by Waksman as "Actin-
 EMI3.2
 clams 16111% Soil Science 8 71-214 (1919) 7 and which he later referred to as Strelptg = ces e = threus-. As will be explained below, there are certain differences between the cultural properties of the organism described by Waksman and those of the new isolated microorganism object of the present invention, so that there is a some doubt as to the proper identification of the isolated microorganism. However, the applicant has provisionally classified the isolated microorganism which is the subject of the present invention.
 EMI3.3
 as being a variety of SttBptomyces erythreus and, therefore, named the organism Stre] 2t = c e s er vthreus, strain M5m12559.



   The microorganisms were isolated from a soil sample from Iloilo City, Iloilo, Philippine Islands; the isolation procedure being as follows: a sample of the soil is suspended in sterile distilled water, the suspension is strongly diluted and a small sample is placed on nutrient agar. The plate is incubated at about 30 ° C. for a week. The tiny, hard, scattered colonies of Streptomyces erythreus are removed with a platinum loop and used to inoculate agar plates to obtain larger amounts of the organism.



   The present invention will be described with particular reference to the aforementioned variety of the organism mentioned above, but it is understood that the fermentation processes which are the subject of the present invention not only include the use of Streptomyces erythreus. , strain M5-
 EMI3.4
 12559, but also that of other erythromycin producing varieties of Streptornxces erythreus, these varieties being readily obtained and isolated by methods commonly applied for the isolation and modification of varieties, which methods include the selection of the varieties. cultured organisms and exposure of the organisms to modifying agents such as x-rays, ultraviolet light and chemical agents, for example nitrogen mustard.

   Examples, given as an indication, of other varieties
 EMI3.5
 erythromycin-producing Stre1: 1tomyces erythreus, strains M5- i464k M5-14642 and M5-146430
Samples of these organisms are available from the Research Laboratories of ELI LILLY and COMPANY, Indianapolis, Indiana, United States of America.
 EMI3.6
 



  StreDtonvces erythreus, strain M5-12559 is characterized by the numerous physical, cultural and physiological tests described in the following paragraphs. The system of Ridgway, Color Standards and Nomenclature (1912), is used for the designation of most colors and, when this system is used, the initial letter of the color is a capital letter.
 EMI3.7
 When growing on agar-agar broth, Streutoyces e- rhythmreus, strain M5-12559, forms a branchy mycelium whose spores in series are arranged in straight or irregularly coiled chains but, in general, are not arranged following a separate spiral organization.



  Growth is typically slow, reduced, and ranges from low to moderate amounts on most media, with the relative abundance of vegetative and serial growth varying with the nutrient medium used.



   On a simple synthetic agar-agar medium, the vegetative mycelium is rare and flat. The aerial mycelium is powdery, white and is present in moderate quantities. When the crop ripens, the aerial part turns a pale pink color. A soluble reddish-wine pigment forms and darkens as the culture ages.



   When the organism is cultivated on agar-agar media containing complex sources of nitrogen, the vegetative mycelium is relatively more abundant than the aerial part. Vegetative growth is typical-

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 ment high and has a tendency to penetrate the agar-agar. In such cultures, the aerial mycelium is usually sparse and white. In addition to the soluble reddish-wine pigment, a brown pigment occurs on many complex media.



   The body is physiologically very active. Starch and gelatin are easily hydrolyzed although the casein in "sunflower milk" cultures is not attacked. Most of the common carbon sources are commonly used for growth. Temperatures of about 30 to 37 C appear to be best for growth and sporulation.



   MICROSCOPIC MORPHOLOGY.



   The morphology of streptomyces erythreus, strain M5-12559, after it has grown on synthetic agar-agar and agar-agar containing calcium malate is given below: Agar -synthetic agar:
There is an aerial mycelium which ramifies moderately with spores on conidiophores in short, rectilinear or irregularly coiled and curved strings, but the typical spiral type. The spores are ovoid and their dimensions range from about 0.5 x 0.8 to about 0.8 x 1.0. The gram stain reaction is positive.



  Calcium malate agar.



   The observed microscopic morphology is the same as that obtained when growing the organism on synthetic agar, except that the spore strings are considerably more hooked and coiled, but not in regular spirals.



     CULTURAL CHARACTERISTICS.



   The cultural characteristics of the organism in a number of common media are given in the following paragraphs: Synthetic agar.



    Moderate vegetative development; cream color and darkening due to the intense color of the soluble pigment; reduced growth of white aerial mycelium which provides a pale tawny reflection of wine as the crop matures; moderate sporulation observed under the microscope; production of soluble pigment more pronounced than in other media, this pigment being reddish-wine-colored in young cultures and turning dark wine-brown or almost black after long incubation; of 4-6 mm. in diameter with raised centers and flat, wavy edges; aerial mycelium thin, spreading, filamentous.



  Glucose and asparagine agar.



   Colorless vegetative growth, rare, with limited growth of aerial mycelium of white color to pale pinkish cinnamon formed only after long incubation; sparse sporulation by microscopic observations; soluble pigment of light ocher salmon color; 2 to 3 mmo in diameter, irregularly convex with complex or wavy edge; smooth or wrinkled surface with a powdery texture.



  Emerson's Agar.



   Good vegetative growth; cream reverse turning cameo brown after 14 days incubation and hay red after 28 days; this in a thin layer of white aerial mycelium; soluble pigment dark hazelnut color; colonies of 3 to 5 mm. in diameter with wrinkled and convoluted surface, raised or sagging centers and irregular wavy edges; colony morphology variable, some lacking aerial mycelium and others covered with a powdery spore crust.

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  Calcium malate agar.



   Colorless, sparse vegetative growth with moderate amounts of pale pink cinnamon aerial mycelium; moderate sporulation by microscopic observation; soluble reddish-red wine, fungus to army brown after prolonged incubation; colonies of 1 to 3 mm. in diameter with raised centers and filamentous edges; fine, powdery textured surface spores; microscopically granular hale of very rare aerial mycelium just outside the periphery of the colony.



  Glucose agar.



    Moderate vegetative growth; dark olive buff color turning tan "Rood" brown; white aerial mycelium in moderate quantity; soluble pigment of "Rood" brown color resembling that formed on Emerson's agar-agar, but less dark; colonies of 2 to 4 mm. in diameter, raised, irregularly convoluted with wavy edge; powdery surface.



  Nutrient agar.



   Rare vegetative mycelium; buff colored reverse; white aerial mycelium in moderate quantity; no soluble pigment; colonies small, reduced 2 to 3 mm. in diameter, irregularly convex with full edge and powdery surface. Larger colonies often have a starry depression in the center.



  On potato.



   Abundant vegetative mycelium with abundant gray-beige aerial mycelium and formation of an almost black upper part gradually darkening to reddish brown at the base.



  Gelatin.



   Moderate growth of white colonies with almost complete hydrolysis in 14 days and complete in 20 days at 25 Co, light brown soluble pigment.



  Glucose broth.



   Uniform skin growth with moderate, white aerial mycelium formed subsequently; soluble pigment almost red ocher.



  Nutrient broth.



   Poor growth, as a few scattered colonies, some with white aerial mycelium; low amount of pearl sediment and no soluble pigment.



  Tyrosine broth.



   Poor growth, flaky; submerged with trace of pearly surface mycelium and no soluble pigment.



   PHYSIOLOGICAL CHARACTERISTICS.



  "Sunflower milk"
In 30 C "sunflower milk" growth is poor and occurs as a ring on the wall of the tube. No hydrolysis or peptonization occurs, but a slow development of an alkaline reaction (14 days).



   At 37 Ci growth is similar to that at 30 C, but the growth and changes are less pronounced.



  Starch agar.



   Starch is strongly hydrolyzed.



  Cellulose (filter paper strips)
There is no growth with ammonium ions or nitrate ions as sources of nitrogen.

 <Desc / Clms Page number 6>

 



   The following tests in which carbon and nitrogen are used were carried out according to the method described by Pridham and Gottlieb; J. Bacteriology, 56, 107-114 (1948).



  Use of carbon sources for growth. a) Commonly used substrates include the following:
1 (+) arabinose -d (+) raffinose starch
1 (+) rhamnose inulin sodium acetate d (-) fructose mannitol sodium citrate d (+) galactose d (-) sorbitol sodium malate d (-) glucose i (-) inositol sodium succinate d (+) maltose dextrin asparagine sucrose b) Poorly used substrates include: d (+) gylose and- salicin.



   It seems that little or no d (+) lactose, dulcitol, sodium formate or sodium tartrate is used. a) Commonly used substrates include: ammonium sulfate and ammonium nitrate. b) Sodium nitrite is not used.



   It is understood, of course, that the above tests for the use of energy sources are carried out under special growing conditions and that the fact that actinomycete does not use certain sources of energy in test conditions do not prohibit their use under different conditions.



   Table II mentions some differences between Actinomy-
 EMI6.1
 these 161 described by Waksman and the StreDtomvces¯¯ethteus, shower M57-12559.



  TABLE II.
 EMI6.2
 Medium Strain M ï2 Act name these 161
 EMI6.3
 
<tb> Morphology <SEP> Microscopic
<tb>
<tb> Number <SEP> of <SEP> spores <SEP> moderate <SEP> Numerous <SEP> spirals <SEP> open
<tb>
<tb> on <SEP> conidiophores <SEP> in <SEP> tes
<tb>
<tb> short <SEP> rosaries, straight <SEP>
<tb>
<tb> or <SEP> irregularly <SEP> running
<tb>
<tb> bés <SEP> and <SEP> rolled up, <SEP> not <SEP> in
<tb>
<tb> <SEP> typical <SEP> spirals (14 <SEP> days)
<tb>
<tb>
<tb> <SEP> cultural characteristics
<tb>
<tb>
<tb> Agar-agar <SEP> Vegetative <SEP> growth <SEP> and <SEP> Vegetative <SEP> growth <SEP> stage
<tb>
<tb> synthetic <SEP> aerial <SEP>:

   <SEP> moderate <SEP> to <SEP> good, <SEP> good, <SEP> is <SEP> developing <SEP> pro
<tb>
<tb> Mycelium <SEP> aerial <SEP> white <SEP> of- <SEP> foundation <SEP> in <SEP> the middle <SEP>.
<tb>
<tb> coming <SEP> partially <SEP> rosâ- <SEP> Mycelium <SEP> aerial <SEP> thick,
<tb>
<tb> be <SEP> during <SEP> of <SEP> the <SEP> maturation;

   <SEP> full, <SEP> white. <SEP> Pigment
<tb>
<tb> <SEP> ranges of <SEP> color <SEP> appear- <SEP> soluble <SEP> Purple <SEP> garnet
<tb>
<tb> sant <SEP> from <SEP> fawn <SEP> binds <SEP> from <SEP> Apple <SEP> turning <SEP> later <SEP>
<tb>
<tb> wine <SEP> Pale <SEP> to <SEP> Salmon <SEP> to <SEP> Bordeaux. <SEP> By <SEP> trans-
<tb>
<tb> Ocré <SEP> clear, <SEP> ferts <SEP> repeated, <SEP> the <SEP> pigment
<tb>
<tb> Pigment <SEP> soluble <SEP> Roux <SEP> binds <SEP> becomes <SEP> color <SEP> binds <SEP> of <SEP> wine.
<tb>
<tb> of <SEP> wine <SEP> becoming <SEP> Brown <SEP> binds
<tb>
<tb>
<tb> of <SEP> wine <SEP> or <SEP> almost <SEP> Black
<tb>
<tb> after <SEP> long <SEP> incubation.
<tb>
 

 <Desc / Clms Page number 7>

 
 EMI7.1
 
<tb>



  Gelatin <SEP> Soluble <SEP> pigment <SEP> brown <SEP> rare. <SEP> None <SEP> soluble <SEP> pigment.
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>



  Agar-agar <SEP> with <SEP> malate <SEP> Pigment <SEP> soluble <SEP> Roux <SEP> binds <SEP> None <SEP> pigment <SEP> soluble
<tb>
<tb>
<tb>
<tb>
<tb> of <SEP> calcium <SEP> of <SEP> wine <SEP> going <SEP> sinking <SEP> until
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Brown <SEP> Army <SEP> after <SEP> long
<tb>
<tb>
<tb>
<tb>
<tb> incubation.
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>



  "Sunflower <SEP> milk <SEP>" <SEP> Poor growth <SEP>; <SEP> au- <SEP> Growth <SEP> in <SEP> zones <SEP> of
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> cune <SEP> hydrolysis <SEP> or <SEP> pepto- <SEP> surface <SEP> yellowish; <SEP> very
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> nization. <SEP> Slow <SEP> development <SEP> coagulation <SEP> (no
<tb>
<tb>
<tb>
<tb>
<tb> slow <SEP> from <SEP> the <SEP> reaction <SEP> alka- <SEP> complete <SEP> in <SEP> 50 <SEP> days).
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> line.
<tb>
 



   As noted above, Streptomyces erythreus, strain M5-12559, can be grown in a culture medium to obtain an effective antibiotic agent. The culture medium can be any of a number of culture media because, as is apparent from the use tests described above, the organism is likely to use many. sources of energy. However, in view of economy of production, maximum yield of antibiotic and ease of isolation of erythromycin, certain culture broths are preferable. Thus, for example, the presently preferred sources of the carbohydrate present in the culture broth are starch and glucose.

   It is also possible to use other sources such as sucrose, dextrin, molasses, etc.



  The preferred sources of nitrogen are the product called "corm steep liquor" (steep liquor in a sulphurous water of corn grains); soybean meal and soluble distillates, but other useful sources include casein, amino acid mixtures, peptones (both meat and soybean) etc. Inorganic nitrogen sources such as nitrates or ammonium salts can also be used.



   The inorganic nutrient salts which are incorporated into the medium include the usual salts capable of producing ions, sodium, potassium, calcium, chlorine phosphate, sulphate; etc.



   It is necessary for the growth and development of other microorganisms that traces of essential elements are also present in the culture broth to grow the actinomycete used in the present invention. Such traces of elements are present in the form of impurities resulting from the addition of other constituents of the culture medium.



   In order to obtain the maximum growth and development of Streptomyces erythreus, strain M5-12559, the pH of the culture medium before inoculation of the organism should be adjusted to between 6.0 and 7.5, and preferably sets it to about 6.5. It has been observed that during the period of growth of the organism and production of the antibiotic, the medium gradually becomes alkaline and its alkalinity can reach a pH of between about 7.2 and about 8.5 or more. moreover, the final pH depending, at least in part, on the initial pH of the medium, the buffer solutions present in the medium and the length of time the organism is allowed to grow.



   As preferred for the production of other antibiotics in bulk quantities, aerobic immersion culture conditions are the prime conditions for the production of large quantities of erythromycin. For the preparation of limited quantities, a shaker flask and surface culture in bottles can be used. Further, as is well known, when growth takes place in large tanks, it is preferable to use the vegetative form of the organism during inoculation of the production tanks in order to avoid delay. pronounced in the production of the antibiotic and the resulting inefficient use of the equipment.

   Accordingly, it is desirable, first, to produce a vegetative inoculum of the organism by inoculating a relatively small amount of culture medium with the organism in the form of spores and,

 <Desc / Clms Page number 8>

 after obtaining a young and active vegetative inoculum, transfer the vegetative inoculum aseptically into large tanks. The medium in which the vegetative inoculum is produced may be the same medium as that used for the production of the antibiotic or a different medium.



   Streptomyces erythreus. strain M5-12559, grows well at temperatures between about 25 C and about 37 Co Optimum antibiotic production appears to occur when the culture broth is maintained at about 26-30 Co
As usual, in the production of antibiotics by immersion culture methods, sterile air is injected into the culture medium. In order to achieve efficient growth of the organism and production of the antibiotic, the volume of air used in the tank production of erythromycin is preferably greater than 0.1 volume of air per minute. and by volume of culture medium.

   More efficient growth and production of the antibiotic is ensured when the volume of air used is at least 0.4 volumes of air per minute per volume of culture broth.



   The magnitude of the production of the antibiotic and the concentration of antibiotic activity in the culture medium during the period of growth of the microorganism can be easily followed by test samples of the broth. culture by determining their antibiotic activity on organisms known to be sensitive to the antibiotic, for example, Staphylococcus aureus and Mycobacterium tuberculosis. For such determinations it is advantageous to use the assay which consists of serially diluting culture samples, adding parts of the culture samples, adding parts of the samples diluted with nutrient agar. in fusion, to solidify the agar-agar in a Petri dish; to i- noculate the plaque with a young culture of S. aureus or M.

   Tuberculosis and to determine which is the greatest dilution of the culture medium which causes the complete inhibition of the growth of the organism on the nutrient agar.



   The production of the antibiotic can also be followed by turbidimetric assay processes such as those commonly used in the production of other antibiotics.



   In general, the maximum production of the antibiotic after inoculation of the culture broth occurs in about 2 to 5 days when using an aerobic submerged culture and in about 5 to 10 days when using the surface culture or in shaker flasks.



   The antibiotic compounds which are the subject of the present invention can be recovered from the culture broth by extraction or adsorption techniques. The former are preferred for industrial production since they require less time and are less expensive.



  For the extraction of the antibiotic compound contained in the culture medium, preferred are water-immiscible, polar and organic solvents, such as fatty acid alkyl esters; for example ethyl acetate and amyl acetate; chlorinated hydrocarbons, for example chloroform and ethylene dichloride; alcohols having slight solubility in water, for example butanol and amyl alcohol; ketones showing slight solubility in water, for example methyl amyl ketone and ethers, for example ethyl ether and dibutyl ether. Other solvents of a similar nature can also be used.



   To obtain the antibiotic in its crude form, the antibiotic material contained in the organic solvent extract consisting of the culture broth can be evaporated to dryness, preferably in vacuo.



  Alternatively, the antibiotic material can be separated from the culture broth by contacting the filtered broth with an absorbent agent.



  Absorbents can also be effectively used for purification by adsorption chromatography using absorbents.

 <Desc / Clms Page number 9>

 such as activated alumina, silica gel, magnesium aluminum silicate, etc. The antibiotic is easily eluted from the adsorbent using a polar organic solvent in which the compound. antibiotic is soluble. Activated charcoal is not particularly well suited for use in a chromatographic column since charcoal strongly adsorbs the antibiotic so that the adsorbed material is eluted only with difficulty.

   However, activated charcoal can be used successfully to adsorb the antibiotic directly from the culture broth if the charcoal is pre-coated with an agent, for example acetic acid in order to decrease the binding affinity of the broth. charcoal for the antibiotic.



   When using an extraction process alone to recover the antibiotic, a suitable method of recovering the antibiotic from the extraction solvent involves evaporating the solvent to a relatively small volume and precipitating it. the antibiotic of the solvent by the addition of a miscible solvent in which the antibiotic has a slight
 EMI9.1
 solubility. The antibiotic material which precipitates in the crude state, but solid, and sometimes even in the crystalline form, is then purified again by recrystallization from one or more solvents or mixtures thereof. Suitable solvents for recrystallization are aqueous acetone, aqueous methanol, etc.
 EMI9.2
 



  A presently preferred way to isolate erythromyonia in the form of its base is to alkaline the filtered culture broth to a pH of about 9 to a pH of about 10, preferably 9.5 and extract the filtered broth. broth adjusted with an alkyl acetate, for example amyl acetate. The erythromycin base is then extracted which dissolves
 EMI9.3
 in acetate d-laiive in water adjusted to a pH below 6.5, preferably a pH of about 5. The volume of the aqueous extract is reduced, for example by evaporation in water. vacuo to initiate the precipitation and we make it in-
 EMI9.4
 Following alkaline to a pH of about 9.5, after which the erythromveine base separates out in the solid form, usually crystalline.

   The base is isolated by filtration or centrifugation and purified by recrystallization.
 EMI9.5
 



  Acid addition salts of erythromycine can be obtained by treating an aqueous solution of erythromyony base with one equivalent of an acid and evaporating the solution in vacuo to dryness. Alternatively, an erythromycine base dissolved in an organic solvent can be treated with the acid or its solution and the erythromycine salt can be precipitated directly from the solution. When preparing acid addition salts of strong acids, care should be taken during the addition of the acid to the antibiotic to avoid high local concentrations of
 EMI9.6
 acid since erythromycin is relatively unstable in low pH solutions.

   By way of illustrative examples of the salts which can be prepared, there may be mentioned the hydrochloride, the sulfate, the citrate, the mandelate, the oleate, the palmitate, the myristate, the stearate and the oxalate. Other similar salts can easily be prepared by the processes just mentioned. For therapeutic use, the salts chosen should obviously be relatively non-toxic.



   When the culture broth is extracted or treated with an adsorbent having a lower alkalinity corresponding to a pH of about 9, the isolated antibiotic compound is generally an erythromycin complex.



  The complex shows the characteristic antibiotic properties of the base
 EMI9.7
 of eryi; 1ir omy cine, but exhibits greatly reduced solubility in water and, in general, lower solubility in organic solvents.



  Erythromycin base can be obtained from the complex by dissolving or suspending the complex in water by adjusting the pH of the medium.
 EMI9.8
 about 9.8 aqueous mixture to separate the erythromycine base from the complex and extracting the alkaline mixture with an organic solvent.



  The erythromycin base is isolated which passes from water into the organic solvent and purified by recrystallization.

 <Desc / Clms Page number 10>

 



   The base which is constituted by erythromycin has the following physical and chemical properties:
Erythromycin crystallizes in white needles which melt with prior softening at about 136-1400C. on a Köfler block for micro-determination of the melting point. It is soluble at about 2 mmg per cm 3 of water and is very soluble in alcohol, acetone, chloroform, acetonitrile and ethyl acetate. It is moderately soluble in ether, ethylene dichloride and amyl acetate.



   A potentiometric determination in a solution of dimethylformamide in water (in a proportion of 2/1 by volume) reveals the presence of a group capable of being determined of pK [alpha] '= 8.8.



   The molecular weight determined by the assay data appears to be around 725.



   Erythromycin crystallizes from aqueous acetone as needles and short stems before positive elongation and parallel extinction. Samples of crystals dried at room temperature under vacuum over phosphorus pentoxide show the following refractive indices in monochromatic sodium light at 27 C. n1 = 1.452 n2 = 1.473
The specific rotation of a sample dried at room temperature under vacuum over phosphorus pentoxide for 4 hours is written as follows: [[alpha]] 25 = 78 D c = 1.99% (volume weight) in ethanol.



   An average of several elementary analyzes of samples which, before analysis, were dried at 60 ° C. under vacuum for 3 hours over phosphorus pentoxide gives the following values:
 EMI10.1
 
<tb> Carbon <SEP> 60.40 <SEP>% <SEP>
<tb>
<tb> Hydrogen <SEP> 9, <SEP> 26 <SEP>%
<tb>
<tb> Nitrogen <SEP> 2.07 <SEP>% <SEP>
<tb>
<tb> Oxygen <SEP> 28,27 <SEP>% <SEP>
<tb> (by <SEP> difference)
<tb>
 
A sample of material dissolved at a concentration of about 3.14% in chloroform (weight by volume) gives the following visible bands in an infrared spectrum over the range 2.4 to 12.0:

   2.82, 3.32, 5.77, 5.91, 6.86, 7.13, 7.25, 7042, 7.78, 8.02, 8.54, 8.98, 9.16, 9.32, 9.49, 9.86, 10.23, 10.42, 1009, 11.17, 11055 and Il. 9.



   An x-ray diffraction pattern of the powdered product using unfiltered chromium radiation and a wavelength value of 2.2896 A for the calculation of interplanar spaces gives the following values:
 EMI10.2
 
<tb> "d" <SEP> (Interplanar <SEP> spacing) <SEP> 1/11 <SEP> (Relative <SEP> intensity)
<tb>
<tb>
<tb> 14.5 <SEP> 0.63
<tb>
<tb> 13.3 <SEP> 1.00
<tb>
<tb>
<tb> 10.9 <SEP> 0.06
<tb>
<tb>
<tb> 9.9 <SEP> 0.38
<tb>
<tb>
<tb> 8.65 <SEP> 0.25
<tb>
<tb>
<tb> 7.70 <SEP> 0.38
<tb>
<tb>
<tb> 7.10 <SEP> 0.13
<tb>
<tb>
<tb> 6.65 <SEP> 0.13
<tb>
<tb>
<tb> 5.80 <SEP> 0025
<tb>
<tb>
<tb> 4.95 <SEP> 0.13
<tb>
<tb> 4.65 <SEP> 0.06
<tb>
 

 <Desc / Clms Page number 11>

 
The absorption spectrum in the ultra-violet is characterized by a single broad peak of low intensity before a maximum of 280 m for a pH of 6.3,

  # (molar extinction) being about 50.



   When recrystallized from petroleum ether or from a mixture of water and acetone, erythromycin produces fine needles crystallized in the hexagonal system.



   Erythromycin hydrochloride has the following = physical and chemical properties:
It crystallizes in aqueous solvents in the form of white needles which melt at about 170-173 ° C on a Köfler block for micro-determination of the melting point. It is very soluble in water and lower alcohols and it is slightly soluble in ethyl acetate. It is only very slightly soluble in solvents such as ether, amyl acetate, chloroform and the like.



   An average of several elemental analyzes of samples of erythromycin hydrochloride which, prior to analysis, were dried under vacuum at 78 ° C. for 3 hours over phosphorus pentoxide gives the following values:
 EMI11.1
 
<tb> Carbon <SEP> 58.74 <SEP>%
<tb>
<tb> Hydrogen <SEP> 8.89 <SEP>%
<tb>
<tb> Nitrogen <SEP> 1.89 <SEP>%
<tb>
<tb> Chlorine <SEP> 4.58 <SEP>%
<tb>
<tb> Oxygen <SEP> (by <SEP> difference) <SEP> 25.90 <SEP>%
<tb>
 
A sample of erythromycin hydrochloride digested in petroleum ether gives the following visible bands in an infrared spectrum over a range of 2.4 to 12.0: 2.94. 3, 38. 5.79. 5.88. 6.85. 7.26.



  7.43.7.78. 7.88. 8.01. 8.55.9.03. 9.24. 9.46. 9.92. 10.47. He, 15. 11.5 and 12.0.



   The erythromycin complex is identified by the following physical and chemical properties:
The complex crystallizes as white needles which melt at about 82-83.5 C (uncorrected) as measured using a Fisher-Johns melting point apparatus. It is quite soluble in acetone, lower alcohols; chloroform and acetylene dichloride, but it is only slightly soluble in higher ketones and ethers.



   Potentiometric determination of the base in a solution of dimethylformamide in water (parts by volume: 2: 1) reveals the presence of ionizable groups of pkD [alpha] 7.6 and pkD [alpha] '8.4.



   The molecular weight as determined by x-ray crystallography data using a sample dried in air at room temperature under vacuum over phosphorus pentoxide is about 1130.



   The specific rotation index of a sample dried under vacuum at room temperature over phosphorus pentoxide is as follows: [[alpha]] 25 = -47 D c = 2% (volume weight) in ethanol .



   An average of several elementary analyzes of samples of erythromycin complexes which, prior to analysis, have been dried for 12 hours at room temperature under vacuum over phosphorus pentoxide gives the following values:

 <Desc / Clms Page number 12>

 
 EMI12.1
 
<tb> Carbon <SEP> 64.71 <SEP>% <SEP>
<tb>
<tb> Hydrogen <SEP> 10.23 <SEP>% <SEP>
<tb>
<tb> Nitrogen <SEP> 1.35 <SEP>% <SEP>
<tb>
<tb> Oxygen
<tb> (calculated <SEP> by <SEP> difference <SEP> 23.71 <SEP>%
<tb>
 
A sample of the complex dried under vacuum at room temperature and dissolved to a concentration of about 5.53% in chloroform (weight-to-volume) gives the following visible bands in an infra-red spectrum over the range from 2.8 to 12.0:

     2.85. 3.40. 3.49. 5.79. 6.25. 6.86. 7.14.



    7.26. 7.44. 7.80. 8.05. 8.54. 8.98. 9.21. 9.49. 9.88. 10.18. 10.43. He, 04.



  11.16. 11.52 and 11.9.



   An X-ray diffraction diaphragm of the powdered product, using unfiltered chromium radiation and a wavelength value of 2.2896 A for the calculation of interplanar spacings, gives the following values:
 EMI12.2
 
<tb> "d" <SEP> (Interplanar <SEP> spacing) <SEP> I / II <SEP> (Relative <SEP> intensity)
<tb>
<tb>
<tb>
<tb>
<tb> 21.2 <SEP> 0.13
<tb>
<tb>
<tb>
<tb> 19.0 <SEP> 1.00
<tb>
<tb>
<tb>
<tb>
<tb> 16.7 <SEP> 0.03
<tb>
<tb>
<tb>
<tb>
<tb> 15.3 <SEP> 0.13
<tb>
<tb>
<tb>
<tb>
<tb> Il, 0 <SEP> 0.05
<tb>
<tb>
<tb>
<tb>
<tb> 10.4 <SEP> 0.13
<tb>
<tb>
<tb>
<tb>
<tb> 10.1 <SEP> 0.13
<tb>
<tb>
<tb>
<tb>
<tb> 9.53 <SEP> 0.11
<tb>
<tb>
<tb>
<tb>
<tb> 8.25 <SEP> 0.08
<tb>
<tb>
<tb>
<tb>
<tb> 7.86 <SEP> 0, <SEP> 03 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> 7.57 <SEP> 0.13
<tb>
<tb>
<tb>
<tb>
<tb> 7.18 <SEP> 0,

  13
<tb>
<tb>
<tb>
<tb>
<tb> 6.92 <SEP> 0.13
<tb>
<tb>
<tb>
<tb>
<tb> 6.56 <SEP> 0, <SEP> 03 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> 6.30 <SEP> 0.03
<tb>
<tb>
<tb>
<tb>
<tb> 6.20 <SEP> 0.21
<tb>
<tb>
<tb>
<tb>
<tb> 5.89 <SEP> 0, <SEP> 03 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> 5.65 <SEP> 0.05
<tb>
<tb>
<tb>
<tb>
<tb> 5.36 <SEP> 0.05
<tb>
<tb>
<tb>
<tb>
<tb> 5.17 <SEP> 0.11
<tb>
<tb>
<tb>
<tb>
<tb> 5.00 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 4.86 <SEP> 0.21
<tb>
<tb>
<tb>
<tb>
<tb> 4.74 <SEP> 0.05
<tb>
<tb>
<tb>
<tb>
<tb> 4.69 <SEP> 0, <SEP> 05 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> 4.52 <SEP> 0.05
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 4.42 <SEP> 0.21
<tb>
<tb>
<tb>
<tb> 4.28 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 4.11 <SEP> 0.08
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 4.01 <SEP> tf
<tb>
<tb>
<tb>
<tb> 3.87 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 3,

  77 <SEP> tf
<tb>
<tb>
<tb>
<tb> 3.71 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 3.58 <SEP> 0, <SEP> 03 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> 3.45 <SEP> 0, <SEP> 03 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> 3.28 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 3,19 <SEP> tf
<tb>
<tb>
<tb>
<tb> 3, the <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 2.73 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 2.63 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 2.59 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 2.52 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 2,49 <SEP> tf
<tb>
<tb>
<tb>
<tb>
<tb> 2.34 <SEP> tf
<tb>
<tb>
<tb>
<tb> z34 <SEP> (tf <SEP> means <SEP> very <SEP> weak) -tif
<tb>
 

 <Desc / Clms Page number 13>

 
The ultraviolet absorption spectrum of an ethanol solution of the complex shows a maximum at 274 m
1%
E = 1.07
1 cm.



   When recrystallized from petroleum ether or from a mixture of water and acetone, the complex gives fine needles crystallized according to the hexagonal system which show parallel extinction and positive elongation, the elongation being parallel. to axis c. At 25 C in monochromatic sodium light, the refraction indices of the crystals are as follows: = 1.509 w = 1.499
The sign of double refraction is positive.



   Aqueous solutions or suspensions of the antibiotic compounds of this invention are moderately stable at room temperature in the pH range of from about 5.0 to 8.5. For a pH lying outside the above-mentioned range, the antibiotic activity soon ceases to show itself. Even stronger acidic solutions lead to a rapid loss of antibiotic activity and more basic solutions result in a loss that is as certain, if not less rapid. The maximum stability of the solutions of the antibiotic compounds is obtained when the pH of the solutions is maintained between 6 and 8.



     Erythromycin and its salts generally contain amounts of water when crystallized in solvents containing water and they may contain amounts of alcohols or other similar solvents when crystallized from solutions which contain it. The amount. of water or other solvent present appears to depend, at least in part, on the conditions under which crystallization occurs and the degree of drying to which the material is subjected and it can vary from a small percentage up to 'at a high percentage
The invention will be better understood on reading the examples given below by way of illustration only.



   EXAMPLE 1.
 EMI13.1
 



  Preparation of err5jtthromycin:
An inoculum broth is prepared, the composition of which is as follows:
 EMI13.2
 
<tb> Starch <SEP> 14,500 <SEP> kg
<tb>
<tb> <SEP> soy flour <SEP> <SEP> 14,500 <SEP> kg
<tb>
<tb>
<tb> Solid materials <SEP> from <SEP> from
<tb>
<tb> soaking <SEP> of <SEP> grains <SEP> of <SEP> but <SEP> 4,500 <SEP> kg
<tb>
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 4,500 <SEP> kg.
<tb>
<tb>
<tb>



  Calcium <SEP> carbonate <SEP> <SEP> 2,700 <SEP> kg.
<tb>
<tb>
<tb> Water <SEP> 945 <SEP> liters
<tb>
 
The broth is placed in an iron tank with a capacity of 1323 liters and sterilized by heating under pressure at a temperature of about 120 ° C. for 30 minutes. The sterilized broth is cooled and inoculated aseptically with spores of Streptomyces erythreus, strain M5-12559. The organism thrives in the broth at about 26 C. for 45 hours. During the growth period, the broth is stirred and aerated with sterilized air at a rate of about 0.5 vol air per volume of culture broth per minute.



   In a 6048 liter iron tank, a fermentation broth is introduced before the following composition:

 <Desc / Clms Page number 14>

 
 EMI14.1
 
<tb> Starch <SEP> 69,300 <SEP> kg.
<tb>
<tb>
<tb>



  Soybean <SEP> <SEP> <SEP> 69,300 <SEP> kg
<tb>
<tb>
<tb>
<tb> Solid <SEP> <SEP> materials from <SEP> from
<tb>
<tb>
<tb> soaking <SEP> of <SEP> grains <SEP> of <SEP> but <SEP> 23,100 <SEP> kg
<tb>
<tb>
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 15 <SEP> kg
<tb>
<tb>
<tb>
<tb> <SEP> Sodium <SEP> <SEP> 23,100 <SEP> kg
<tb>
<tb>
<tb> Water <SEP> 4.535 <SEP> liters
<tb>
 
The culture broth is sterilized by heating it under pressure at about 120 ° C. for about 30 minutes. The broth is cooled and the above inoculation culture is added aseptically. The organism is allowed to develop in the broth for 4 days at a temperature of 36 C.

   During the growth period, the broth is stirred and injected with sterile air at the rate of about 0.5 volumes of air per volume of broth per minute. At the end of the growth period, the broth shows antibiotic activity equivalent to about 150 mcg. erythromyoin per cm3 of broth. The pH of the culture broth (approximately a volume of 4160 liters) is adjusted to 9.5 with 40% caustic soda solution and filtered to remove the mycelium, the filtration being aided by means of 3% of "Hyflo Super. -Cel "(a filtration promoting product sold by the Johns Manville Company). The clear filtrate is extracted with amyl acetate in a Podbielniak extraction vessel using 1 volume of amyl acetate per 6 volumes of clarified broth.

   The amyl acetate extract is in turn extracted batchwise with water brought to a pH of about 5 by addition of sulfuric acid. Two extractions are carried out, the first with 1/2 volume and the second with 1/4 volume of water brought to a pH of 5 with sulfuric acid. The aqueous extracts are combined and their pH adjusted to 8.0 with sodium hydroxide solution. The alkaline solution is concentrated in vacuo to bring it to a volume of about 113 liters, then the pH of the solution is added to 9.5 with the addition of aqueous caustic soda and allowed to stand. Erythromycin separates out as crystalline material. The crystals are filtered off, the pH of the mother liquor is adjusted to about 8 by adding dilute sulfuric acid and concentrated in vacuo to a volume of about 76 liters.

   We settle. the pH of the solution to about 9.5 and allowed to stand, after which an additional amount of erythromycin separated out as crystals.



  The total amount of erythromycin obtained is approximately 256 gr.



   Erythromycin is purified by several recrystallizations from aqueous acetone (mixture in a proportion of 2: 1). EXAMPLE 2.



   Preparation of erythromycin hydrochloride
1 gr is suspended. erythromycin in 10 cm3 of water and dilute hydrochloric acid is added to bring the pH of the mixture to 6.5. The aqueous solution is concentrated in vacuo to about 1/10 of its original volume and cooled in a refrigerator, after which the erythromycin hydrochloride precipitates in crystalline form. The hydrochloride is purified by recrystallization from a mixture of ethanol and ether.



   EXAMPLE 3.



   Preparation of the erythromycin complex.



   An inoculum broth is prepared before the following composition ¯.:.
 EMI14.2
 
<tb>



  -Starch <SEP> 13, <SEP> 600 <SEP> kg
<tb>
<tb> Soy <SEP> <SEP> flour <SEP> 13,600 <SEP> kg
<tb>
<tb>
<tb> Solid <SEP> <SEP> materials from <SEP> from
<tb>
<tb> soaking <SEP> of <SEP> grains <SEP> of <SEP> but <SEP> 4,500 <SEP> kg
<tb>
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 4,500 <SEP> kg.
<tb>
 

 <Desc / Clms Page number 15>

 
 EMI15.1
 
<tb>



  Calcium <SEP> carbonate <SEP> <SEP> 2, <SEP> 700 <SEP> kg
<tb>
<tb> Water <SEP> 945 <SEP> liters
<tb>
 
The broth contained in an iron tank with a capacity of 1323 liters is sterilized by heating under pressure at a temperature of about 120 ° C. for 30 minutes. The sterilized broth is cooled and inoculated aseptically with Streptomyces erythreus spores, strain M5-12559. The organism thrives in the broth at approximately 26 Ce over a period of 35 hours. During the growth period, the broth is stirred and aerated with sterile air at the rate of about one-half volume of culture broth per minute. At the end of the growth period, good growth of the organism is obtained in vegetative form.



   In a 6,048-liter iron vat, a fermentation medium having the following composition is placed:
 EMI15.2
 
<tb> Glucose <SEP> 113.250 <SEP> kg
<tb>
<tb> Starch <SEP> 113.250 <SEP> kg
<tb>
<tb>
<tb> <SEP> soy flour <SEP> <SEP> 212,900 <SEP> kg
<tb>
<tb>
<tb> Solid <SEP> <SEP> materials from <SEP> from
<tb>
<tb> soaking <SEP> of <SEP> grains <SEP> of <SEP> but <SEP> 41,500 <SEP> kg
<tb>
<tb>
<tb> Beer <SEP> yeast <SEP> <SEP> 22,650 <SEP> kg
<tb>
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 9,060 <SEP> kg
<tb>
<tb>
<tb> Sodium <SEP> <SEP> <SEP> 22,650 <SEP> kg
<tb>
<tb>
<tb> Chloride <SEP> of <SEP> cobalt <SEP> 0.112 <SEP> kg
<tb>
<tb>
<tb> Water <SEP> 4.536 <SEP> liters
<tb>
 
The culture broth is sterilized by heating under pressure in the same manner as for the inoculum broth.

   The cooled fermentation broth is then inoculated aseptically with 472.50 liters of the vegetative inoculum culture broth described above and the organism grows in the broth for 4 days at a temperature of 26 C. During the growth period, the growth mixture is stirred and sterile air is passed through it at the rate of 0.5 volumes of air per volume of broth per minute. At different times during the growth period, the amount of antibiotic contained in the culture broth is determined by the method of dilution in agar-agar until, at the end of the growth period of 4 days the amount of antibiotic in the broth reaches an activity equivalent to about 250 mcg of erythromycin per cm3 of broth.

   During the growth period, the pH of the broth changes from its initial value of 6.2 to a value of about 8.0. The antibiotic is isolated from the culture broth as an erythromycin complex by the following procedure. :
The culture broth was filtered on a 36 "Sperry press through 3% of the product" Hyflo-Super-Cel "The filtrate was degreased by extracting it with about 400 liters of petroleum ether. adjust the pH of the extraction filtrate to about 8.5 by adding a sodium hydroxide solution and extracting it with twice 1000 liters of ethyl acetate. The ethyl acetate extracts are combined and mixed. evaporates in vacuo to give about 661 g of a dark brown solid.

   The solid is triturated with petroleum ether to obtain the erythromycin complex as a light tan solid. The solid is dissolved in a minimum amount of ethanol and water is added until the solution becomes cloudy. The cloudy solution was cooled in a refrigerator for about 48 hours to obtain the complex as crystals. The crude crystals were recrystallized from a mixture of alcohol and water, as described above, and recrystallized twice from aqueous acetone.



   The afasi material obtained is an erythromycin complex

 <Desc / Clms Page number 16>

 Sibly pure which melts at approximately 82-83 C.



   EXAMPLE 4.



   Preparation of erythromycin complex in shaker bottle.



   A sporulated culture of Streptomyces erythreus, strain M5-12559 is obtained by growing the organism on a nutrient agar plate. The spores are recovered in the form of an aqueous suspension covering the plate with low amount of water and gently scraping the spores off the surface of the plate.



   About 1 cm3 of the resulting spore suspension is used to inoculate the following sterile culture medium under sterile conditions:
 EMI16.1
 
<tb> Glucose <SEP> 1,5 <SEP> gr.
<tb>
<tb>
<tb>



  <SEP> soy flour <SEP> <SEP> 1,5 <SEP> gr.
<tb>
<tb>
<tb>
<tb>



  Solid <SEP> <SEP> materials from <SEP> from
<tb>
<tb>
<tb> soaking <SEP> of <SEP> grains <SEP> of <SEP> but <SEP> 0, <SEP> 5 <SEP> gr.
<tb>
<tb>
<tb>
<tb>



  Sodium <SEP> <SEP> <SEP> 0.5 <SEP> gr.
<tb>
<tb>
<tb>
<tb>



  Calcium <SEP> <SEP> carbonate <SEP> 0.2 <SEP> gr. <SEP>
<tb>
<tb>
<tb>
<tb> Water <SEP> 100 <SEP> cm3
<tb>
 
The inoculated culture medium is incubated at about 27 ° C. for about 48 hours until good vegetative development is observed. The culture medium containing the vegetative mycelium is divided into 10 equal parts and this is used to inoculate ten 1 liter Erlenmeyer flasks, each of these flasks containing about 200 cm3 of sterilized fermentation culture medium having the following composition:
 EMI16.2
 
<tb> Starch <SEP> 30 <SEP> gr.
<tb>
<tb>



  Soy <SEP> <SEP> flour <SEP> 30 <SEP> gr.
<tb>
<tb>
<tb>



  Solid <SEP> <SEP> materials from <SEP> from
<tb>
<tb> soaking <SEP> of <SEP> grains <SEP> of <SEP> but <SEP> 10 <SEP> gr.
<tb>
<tb>
<tb>



  Sodium <SEP> <SEP> <SEP> 5 <SEP> gr.
<tb>
<tb>
<tb>



  Carbonate <SEP> of <SEP> calcium <SEP> 2 <SEP> gr.
<tb>
<tb>
<tb> Water <SEP> 2 <SEP> liters <SEP>
<tb>
 
We place the vials. containing the inoculated culture media in an incubation chamber maintained at a temperature of about 28 C and shaking for about 80-90 hours on a shaking apparatus having a stroke of 50 mm and operating at a rate of 114 back and forth per minute.



  From time to time, samples of the culture medium are tested for their level of antibiotic activity. When the level of antibiotic activity reaches a value of about 200 mcg / cm3 of broth, the broths are combined from the shaking vials and filtered to remove the mycelium.



   Product showing antibiotic activity as an erythromycin complex is recovered from the broth by extracting the broth twice with equal volumes of butanol and evaporating the butanol to dryness in vacuo. The solid erythromycin complex which remains as a residue is purified by several recrystallizations from aqueous acetone.



   EXAMPLE 5
Purification of the erythromycin complex by adsorption on carbon.



   1 liter of the filtered culture broth obtained by the flask method of Example 4 is treated with 2 g of "Norit SG" (activated charcoal)

 <Desc / Clms Page number 17>

 treated with acid, prepare by stirring the charcoal in 5 times its weight of 5% acetic acid for 15 minutes, the charcoal filtered off and washed with 25 times its weight of water (dry ). The broth-carbon mixture is stirred for about 1/2 hour, the carbon is separated by filtration and washed with 500 cm3 of water. The activated charcoal is stirred with 500 cm3 of butanol to elute the erythromycin complex. The mixture is filtered to remove carbon, and the butanol filtrate which contains the complex is azeotroped to dryness.

   The erythromycin complex residue is purified by recrystallizing it from aqueous ethanol.



   EXAMPLE 6.



   Purification of the erythromycin complex by chromatography.



     The crude erythromycin as obtained by extraction with ethyl acetate from the culture broth (from Example 3) is dissolved in benzene in an amount of approximately 200 mg of crude complex per 5 cm3. of benzene.



   The benzene solution is passed through a column of silica gel, dimension 19.04 mm x 279 mm. The column is washed with a mixture of 15% methanol in benzene to remove an inactive brown material. The complex is eluted from the column by washing with methanol. The methanol eluate is evaporated to dryness in vacuo and the residue of the erythromycin complex, the purity of which is greater than 90%, is purified by recrystallization from aqueous acetone.



   EXAMPLE 7.



   Preparation of erythromycin in shaker flasks.



   A culture broth exhibiting antibiotic activity is obtained in accordance with the process described in Example 4. The product exhibiting the antibiotic activity of the culture broth is recovered from the broth in the form of erythromycin as follows:
The broth is made alkaline by bringing it to a pH of about 9.8 by adding caustic soda and extraction is carried out with 2 1/4 volume parts of ethyl acetate. The combined ethyl acetate extracts which contain erythromycin are extracted with two parts by equal volume of water acidified with sulfuric acid so as to bring its pH to about 5.0. The aqueous extracts are combined and the pH adjusted to about 7.0 with aqueous caustic soda.

   The neutralized solution is reduced to about 1/10 of its original volume until the point of onset of precipitation. The pH of the aqueous mixture is brought to about 9.5 with caustic soda solution and left to stand in the cold for several hours.



  The erythromycin which decants is filtered off and recrystallized from aqueous ethanol.



   EXAMPLE 8.



   Purification of Erythromycin by the Chromatographic Method.



   The pH of an aqueous solution (containing e-rhythmycin such as that obtained according to the process of Example 1 is adjusted to 9.5 by extracting the culture broth filtered with amyl acetate. and the amyl acetate extract with water) with aqueous alkali and extracted with benzene.



   A 100 cc aliquot of the benzene extract having antibiotic activity equivalent to 10 mg erythromycin per cc of benzene is passed through a 25 mm aluminum magnesium silicate ("Florisel") column. in diameter and 304 mm long. The column is washed with 5% methanol in benzene to remove a brown band appearing in the column, and until the eluate becomes substantially colorless. Antibiotic absorbed in the column was removed by elution with 5% methanol in benzene. The eluate is evaporated to dryness in vacuo and the erythromycin residue recrystallized from aqueous acetone.

 <Desc / Clms Page number 18>

 



   EXAMPLE 9.



   Preparation of erythromycin from the erythromycin complex.



   1 g of erythromycin complex is suspended in 100 cm3 of water and the pH of the suspension is adjusted to about 6.3 by adding dilute hydrochloric acid. The aqueous mixture is extracted with dilute chloroform and the pH of the extracted solution is adjusted to about 9.8 with aqueous caustic soda. The alkaline solution is extracted twice with equal parts by volume of amyl acetate, the amyl acetate extracts are combined and evaporated to dryness in vacuo. The basic erythromycin residue is recrystallized from aqueous ethanol.



   EXAMPLE 10.



   Preparation of erythromycin hydrochloride.



   An aqueous solution of an antibiotic such as that described in Example 6 and having an antibiotic activity equivalent to about 525 mcg of erythromycin per cm3 of solution is adjusted to pH 9.5 to 9.5. , using an aqueous solution of caustic soda, and extracted with 15 cm3 of amyl acetate, which makes it possible to obtain a solution before an antibiotic activity equivalent to 14.8 mg erythromycin per cm3 of solution. 0.1 cm3 of ethanol containing 0.0087 cm3 of concentrated aqueous hydrochloric acid is added to the amyl acetate extract. The mixture is cooled in the refrigerator for several hours, after which the erythromycin hydrochloride separates out as substantially pure crystals.



   CLAIMS.



   1. A process for the preparation of erythromycin and its acid addition salts, comprising culturing a strain of Streptomyces ervthreus producing erythromycin, in a culture medium containing assimilable sources of carbohydrate, of erythromycin. nitrogen and inorganic salts, until significant antibiotic activity is obtained in this organism, in this culture medium; then recovering the erythromycin from the culture medium and, if desired, reacting the erythromycin with an appropriate acid to form the corresponding addition salt.


    

Claims (1)

2. The method of claim 1, wherein the culture medium contains carbohydrate, nitrogen and assimilable inorganic salts, and wherein the erythromycin producing organism is cultured under submerged conditions. and aerobic. 3. Process according to claims 1 or 2, according to which the organism is Streptomyces erythreus, strain M5-12559. 4. A method according to claims 1 or 2, wherein the culture medium is maintained at a temperature of about 25-37 C. 5. The method of claim 4, wherein the growth of the organism continues for a period of 2 to 5 days. 6. A process according to claims 1 or 2, wherein the culture broth at a pH of about 9-10 is extracted with a polar organic solvent immiscible with water. 7. A process for preparing erythromycin and its acid addition salts substantially as described above. 8. Erythromycin and its acid addition salts obtained by the process according to the preceding claims. 9. Erythromycin hydrochloride. 10. Erythromycin complex. 11. Antibiotic substance from the group comprising a nitrogenous base and its acid addition salts, said base having the following properties: molecular weight of about 725; pK [alpha] 'of 8.8, giving, when assayed <Desc / Clms Page number 19> in a solution of dimethylformamide in water (in a proportion of 2/1 by volume); and, in a chloroform solution at a concentration of about 3.14% (weight-to-volume), the following visible bands in an infrared absorption spectrum over the range 2.4 to 12 /: 2.82, 3.32, 5.77, 5.91, 6.86, 7.13, 7.25, 7.42, 7.78, 8.02, 8.54, 8.98, 9, 16, 9.32, 9.49, 9.86, 10.23, 10.42, la, 9, Il, 17, Il, 55 and 11.9. 12. Nitrogenous base according to claim 11. 13. Hydrochlorate of the nitrogenous base according to claim 11, said salt, in petroleum ether, giving the following visible bands in an infra-red spectrum over a range from 2.4 to 12: 2.94, 3.38. , 5.79, 5.88, 6.85, 7.26, 7.43, 7.78, 7.88, 8.01, 8.55, 9.03, 9.24, 9.46, 9 , 92, 10.47, 11.15, 11.5 and 120 14. An organic acid addition salt of a base according to claim 11, said salt, in a chloroform solution of a concentration of about 5.53% (w / v), giving the following visible bands in an infrared absorption spectrum, over the range from 2.4 to 12: 2.85, 3.40, 3.49, 5.79, 6.25, 6.86, 7.14, 7.26 , 7.44, 7.80, 8.05, 8.54, 8.98, 9.21, 9.49, 9.88, la, 18, la, ± 3, 11.04, 11.16, 11.52 and 11.9.